In B-cell acute lymphoblastic leukemia (B-ALL), activation of Notch signaling leads to cell-cycle arrest and apoptosis. We aimed to harness knowledge acquired by understanding a mechanism of Notch-induced cell death to elucidate a therapeutically viable target in B-ALL. To this end, we identified that Notch activation suppresses Polo-like kinase 1 (PLK1) in a B-ALL–specific manner. We identified that PLK1 is expressed in all subsets of B-ALL and is highest in Philadelphia-like (Ph-like) ALL, a high-risk subtype of disease. We biochemically delineated a mechanism of Notch-induced PLK1 downregulation that elucidated stark regulation of p53 in this setting. Our findings identified a novel posttranslational cascade initiated by Notch in which CHFR was activated via PARP1-mediated PARylation, resulting in ubiquitination and degradation of PLK1. This led to hypophosphorylation of MDM2Ser260, culminating in p53 stabilization and upregulation of BAX. shRNA knockdown or pharmacologic inhibition of PLK1 using BI2536 or BI6727 (volasertib) in B-ALL cell lines and patient samples led to p53 stabilization and cell death. These effects were seen in primary human B-ALL samples in vitro and in patient-derived xenograft models in vivo. These results highlight PLK1 as a viable therapeutic target in B-ALL. Efficacy of clinically relevant PLK1 inhibitors in B-ALL patient-derived xenograft mouse models suggests that use of these agents may be tailored as an additional therapeutic strategy in future clinical studies.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common single cancer in children, and patients of any age with relapsed B-ALL have poor outcomes (1–3). Treatment strategies and outcomes for B-ALL are partially based on the presence of high-risk genomic aberrations (2, 4). These include presence of the BCR-ABL oncogene, or the so-called Philadelphia chromosome (Ph+; refs. 1, 2). Another high-risk subgroup in both pediatric and adult populations lacks BCR-ABL, but has gene expression profiles similar to Ph+ cases, and is thus referred to as “Ph-like” (5–7). Currently, standard treatment for B-ALL invariably includes cytotoxic chemotherapies that have significant long-term side effects (8). Therefore, treatment strategies that minimize the need for chemotherapy or utilize other targeted antileukemia agents are highly desirable. Thus, the need for effective and targeted therapeutic approaches is critical for both adult and pediatric patients with B-ALL. Despite recent advances in chimeric antigen receptor T cells, CD19-CD3 bi-specifics, and CD22 antibody–drug conjugates, there remains a significant unmet medical need (9–11). We sought to use our knowledge of the apoptotic effects of Notch signaling on B-ALL cells (12, 13) to find a novel therapeutic approach that could be rapidly tested in the clinic.

Normal development of lymphoid cells is dependent on Notch signaling (14). When Notch receptors (Notch1–4) on the surface of a cell are bound by ligand, 2 successive proteolytic cleavages occur, resulting in the release of intracellular Notch (ICN1-4), which subsequently translocates to the nucleus (15). Once in the nucleus, ICN interacts with a number of cofactors to influence transcription of a variety of downstream target genes, including HES1, HEY1, and DTX1 (16). Constitutive activation of Notch signaling leads to T-ALL (17, 18). Conversely, potent activation of Notch signaling exerts proapoptotic effects in B-ALL cells (12, 13).

Inhibitors of the Notch pathway have demonstrated preclinical success and are in clinical trials for malignancies with overactive Notch signaling, such as breast, colorectal, gliomas, and T-cell malignancies (19). In contrast, options for translatable Notch agonists are extremely limited, due largely in part to lack of specificity. Therefore, we aimed to identify clinically actionable targets downstream of Notch that could mimic its proapoptotic effects in B-ALL. In this study, we identified Polo-like kinase 1 (PLK1) as a B-ALL–specific pathway that contributes to the tumor suppressive effect of Notch activation.

PLK1 is a serine/threonine kinase and negative regulator of p53 that mediates mitotic entry, spindle formation, and chromosome segregation (20, 21). Its expression is elevated in solid tumors arising from several anatomic locations, including bladder, melanoma, colorectal, esophageal, and lung (22). In a variety of malignancies, PLK1 knockdown stabilizes p53, resulting in apoptosis (23). In this study, we describe a mechanism by which Notch activation downregulates PLK1, allowing for p53-mediated cell death. Using a clinically relevant PLK1 inhibitor, we demonstrate that inhibition of PLK1 in cell lines and patient-derived xenograft (PDX) models of B-ALL mimics Notch activation, resulting in cell-cycle arrest and apoptosis. Thus, our work supports the use of PLK1 inhibitors in B-ALL.

Cell culture

Pertinent cell line details are summarized in Table 1. All cells were maintained in RPMI1640 medium (GIBCO) containing 10% heat-inactivated FCS (Hyclone) and 1 mmol/L HEPES, 1 mmol/L glutamine, 1 mmol/L sodium pyruvate, and 1 × nonessential amino acids (all, GIBCO). Cell samples from patients with T-ALL or B-ALL were acquired from the Leukemia Tissue Bank at The University of Texas MD Anderson Cancer Center, with approval from the MD Anderson Institutional Review Board.

Table 1.

Leukemic cell lines used in this study

Human leukemia cell linep53 response to irradiationp53 statusBCR-ABL statusSource ATCC
JM1 (B-ALL) Yes Wild type None CRL-10423 
SB (B-ALL) Yes Wild type None CCL-120 
MUTZ5 Yes Wild type Ph-like  
MHH-CALL4 Yes Wild type Ph-like  
Nalm6 (B-ALL) Yes Wild type Ph+ CRL-3273 
Nalm1 (B-ALL) NA Wild type Ph+ CRL-1567 
Sup B15 (B-ALL) NA Wild type Ph+ CRL-1929 
CEM (T-ALL) No Multiple mutation NA CCL-119 
SupT1 (T-ALL) Yes Wild type NA CRL-1942 
Molt4 (T-ALL) Yes Wild type NA CRL-1582 
Jurkat (T-ALL) Weaker Multiple mutation NA TIB-152 
HL60 (AML) No Null NA CCL-240 
Reh Yes Wild type NA CRL-8286 
HS5 NA Bone marrow fibroblast NA CRL-11882 
Human leukemia cell linep53 response to irradiationp53 statusBCR-ABL statusSource ATCC
JM1 (B-ALL) Yes Wild type None CRL-10423 
SB (B-ALL) Yes Wild type None CCL-120 
MUTZ5 Yes Wild type Ph-like  
MHH-CALL4 Yes Wild type Ph-like  
Nalm6 (B-ALL) Yes Wild type Ph+ CRL-3273 
Nalm1 (B-ALL) NA Wild type Ph+ CRL-1567 
Sup B15 (B-ALL) NA Wild type Ph+ CRL-1929 
CEM (T-ALL) No Multiple mutation NA CCL-119 
SupT1 (T-ALL) Yes Wild type NA CRL-1942 
Molt4 (T-ALL) Yes Wild type NA CRL-1582 
Jurkat (T-ALL) Weaker Multiple mutation NA TIB-152 
HL60 (AML) No Null NA CCL-240 
Reh Yes Wild type NA CRL-8286 
HS5 NA Bone marrow fibroblast NA CRL-11882 

NOTE: Fingerprinted positive cell lines with mycoplasma negative status were used within 20 passages from thawing. p53 protein expression in response to γ-irradiation (800 rads) was confirmed by using cytometry (L.S. Golfman, unpublished observation). Wild-type p53 status was verified by sequencing or querying in the COSMIC database and/or searching the literature. NA, not applicable for irradiation/BCR-ABL studies performed.

Notch activation via coculture on HS5 feeder system or with plate-bound ligands

HS5 cells were transduced with GFP-expressing MSCV-based retroviral plasmid MigR1 (as control) or full-length human DLL1, sorted for 100% GFP positivity and plated. B-ALL cell lines, T-ALL CCRF-CEM cells, and secondarily engrafted B-ALL cells from patients were counted and plated (100,000 cells/well) onto the GFP or DLL1-expressing HS5 feeder system. Alternatively, Notch was activated by the plate-bound DLL1 ligand in protein-G–coated 6-well plates. Plates were treated with 1% BSA (Sigma-Aldrich), washed with PBS, and incubated with 1 μg of human ligands fused to IgG Fc domains, that is, DLL1-Fc (ALX-201-765-0025; Enzo Life Sciences), DLL3-Fc, DLL4-Fc (Adipogen), Jagged-1Fc or -2Fc (1277-JG-050 and 1726-JG-050; R&D Systems) or control IgG-Fc (Jackson Laboratory) in 0.1% BSA independently, with rocking, overnight at 4°C.

HES1 induction and PLK1 knockdown

Human cDNA for Notch target gene HES1 was cloned into the MigR1 retroviral vector (12, 24). PLK1 short-hairpin (sh) RNA from a TRIPZ-Inducible Transfection Starter Kit (RHS4741-EG5347; Dharmacon) was cotransfected with second-generation packaging vectors psPAX2 and pMD2 (ratio 1:1:0.5, respectively), using jetPEI transfection reagents according to the manufacturer's protocol, for 72 hours into 293T cells (provided by Dr. Faye Johnson, MD Anderson). Cells were transduced using the following method: Cells (0.1–2 × 106) were plated with 250 to 500 μL of a viral supernatant and 4 to 8 μg/mL of polybrene (Sigma-Aldrich). After centrifugation at 1,000 × g for 90 minutes, cells were incubated at 37°C in 5% CO2 for 3 to 6 hours before addition of fresh complete culture medium. Upon recovery, cells transduced with doxycycline-inducible PLK1 shRNA or nontargeted control were selected against puromycin. To induce PLK1 knockdown, B-ALL cells were exposed to doxycycline (2 μg/mL) for 2 days. Doxycycline-induced red fluorescent protein expression was confirmed by flow cytometry analysis.

siRNA transfection

Oligonucleotide small-interfering RNA (siRNA) targeting human PLK1 (sc36277) or CHFR (sc37567) and siFITC-nontargeting control scramble (sc36869) were purchased from Santa Cruz Biotechnology. Each siRNA (100 nm) was transfected into cells using Lipofectamine 2000 (Invitrogen) along with control siFITC transfection reagent according to the manufacturer's protocol. After 12 hours of siRNA transfection, FITC-positive leukemic cells were sorted for coculture.

RNA extraction and RT-PCR

Total RNA was isolated from the cells and reverse-transcribed using the RNeasy Mini Kit (Qiagen). Prepared RNA was primed with random hexamers to synthesize cDNA using AMV reverse-transcriptase (Amersham) according to the manufacturer's instructions. qRT-PCR was performed using human-specific Taqman probes for HES1 (Hs00172878_m1), Deltex1 (Hs00614837), HEY1 (Hs03005884), PLK1 (Hs00153444_m1), and GAPDH (4333764F); (Applied Biosystems) per the manufacturer's instructions. CHFR cDNA was amplified with the following primers (forward/reverse, 5′-3′): CAGCAGTCCAGGATTACGTGTG and AGCAGTCAGGACGGGATGTTAC (25). The qPCR reactions were performed on an iCycler (Bio-Rad). All values were normalized to the expression of the control GAPDH housekeeping gene using the 2ΔΔCt method.

Western blotting

Cocultured cells were collected and whole-cell lysates were prepared according to established methods utilizing Triton X-100 or RIPA lysis buffer and 1 mmol/L PMSF supplemented with protease inhibitor cocktail (Calbiochem) and phosphatase inhibitor cocktails I and II (Sigma-Aldrich). BCA Protein Assay Kit (Thermo Fisher Scientific) was used to determine protein concentrations. HRP-linked secondary antibodies against rabbit, mouse, and goat IgGs were purchased from Amersham-GE Life Sciences. Fifty micrograms of protein were loaded for Western analyses as described previously (26).

Immunoprecipitation

For immunoprecipitation of PLK1, CHFR, MDM2, pMDM2, and p53, cells were subjected to lysis with 400 μL of HEPES lysis buffer (pH 7.5) containing 50 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L EGTA, 10 mmol/L Na pyrophosphate, 10 mmol/L NaF, 10% glycerol, 1% Triton X-100, and 1.5 mmol/L MgCl2 at 4°C for 30 minutes. Whole-cell lysates were subjected to centrifugation at 15,000 × g for 20 minutes at 4°C and diluted in RIPA immunoprecipitation buffer (500 μL) containing 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 0.5% Nonidet P-40, 0.1% SDS, and 0.1% sodium deoxycholate with 1 × protease inhibitor mixture (Roche Applied Science) and were precleared with 30 μL of a 50% slurry of protein A-Sepharose (Amersham). Supernatants were incubated with normal mouse serum and appropriate antibodies at 4°C for 3 hours. Immunocomplexes were incubated with protein G–coated magnetic beads (53014; Active Motif) for 45 minutes at 4°C. Immunoprecipitation and wash steps were performed by pelleting the magnetic beads on the side of the tube using a magnetic bar for the removal of supernatant. The precipitates were washed 3 times with the lysis buffer at 4°C, resuspended in 30 μL of the SDS sample buffer, and heated at 100°C for 5 minutes. Proteins were then resolved by 10% SDS-PAGE and transferred onto Immobilon-P membranes for Western blot analysis using the specified antibodies.

Antibodies and pharmacologic inhibitors

Antibodies β-actin (A2066; Sigma-Aldrich), cleaved Notch2 (C651.6DbHN; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), HES1 (ab71559; Abcam; TA500014; Origene), Notch-1, -3, and -4 (ab52627, ab23426, and ab184742; Abcam), Jagged1 (sc11376; Santa Cruz Biotechnology), DLL1 (sc73899; Santa Cruz Biotechnology), p53 (DO1; Santa Cruz Biotechnology), FITC anti-p53 antibody (645803; BioLegend), PLK1 (ab17057; Abcam), CHFR (H00055743-M01; Abnova), PAR (4335-MC-100-AC; Trevigen), MDM2 (OP115; Millipore), pMDM2(ser260) (orb129684; Biorbyt LLC; Bioworld Technology, Inc.), phycoerythrin-labeled donkey anti-rabbit IgG antibody (406421; BioLegend), and anti-ubiquitin (U5379; Sigma-Aldrich) were used. PARP1 inhibitor 3ABA (300 μmol/L; Sigma-Aldrich; ref. 27), DMSO, Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT; Sigma-Aldrich), and PLK1 inhibitors BI6727 (volasertib; ref. 28) and BI2536 (29) (Selleck Chemicals) were used.

Ph-like B-ALL engraftment and therapy

B-ALL patient cells were injected into the tail vein of 5- to 6-week-old NSG-SGM3 mice (500,000 per mouse) after sublethal total body irradiation (800 rads). Peripheral blood was drawn from the tail vein at relevant time points and monitored for engraftment via flow cytometry for human CD45 and CD19 expression. When circulating leukemic cells were observed (3 weeks from initial injection), engrafted mice were treated twice weekly for 2 weeks with DMSO or PLK1 inhibitor BI2536 or BI6727. Peripheral blood leukemia burden was monitored beginning with the first treatment, weekly for 4 weeks. At the end of the 4-week monitoring period, mice were euthanized and peripheral blood, femurs, and tibias harvested. Animals were maintained and all animal experiments were performed under Institutional Animal Care and Use Committee with prior approval from the MD Anderson in compliance with the US Department of Health and Human Services guidelines.

Flow cytometry

Cells were stained with antibodies against human PLK1, total p53, and pMDM2 were quantified by flow cytometry (30). For intracellular staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm (554714), stained with antibody for 40 minutes at room temperature with gentle shacking, and run on a FACSCalibur. Annexin V staining was performed using standard methods (550475; BD Biosciences). FlowJo software (FlowJo LLC) was used for analysis.

CyTOF mass cytometry

Metal-tagged antibodies were provided by the MD Anderson Flow Cytometry Core, and samples were stained as described previously (31, 32), and run on a DVS Sciences CyTOF mass cytometer (Fluidigm). Samples were analyzed using Spanning Tree Progression of Density Normalized Events (SPADE) algorithm as described elsewhere (33).

Expression of relevant genes in ALL patient samples

mRNA expression levels in samples from 206 children with ALL (34 T-ALL, 172 B-ALL) were measured on Affymetrix U133A arrays. To identify significant differences, we used linear models for microarray analysis (LIMMA), the empirical Bayes t test implemented in Bioconductor, and the Benjamini–Hochberg method of false discovery rate estimation. Data were deposited at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE26366.

Statistical analysis

Differences between treatment groups were analyzed by the Student t test or multiple measures ANOVA to assess statistical significance. All in vitro experiments were performed in triplicate unless otherwise noted.

All 4 Notch receptor homologs are expressed across B-ALL subtypes

Previous work from our laboratory (12, 13) demonstrated that Notch signaling results in growth arrest and apoptosis in B-cell malignancies. To better understand the expression of Notch receptors on B-ALL, we evaluated surface expression of Notch1 to Notch4 in patient samples with various Ph status by flow cytometry. Expression of Notch2 was consistently highest, whereas the other receptors were present at lower and variable levels (Fig. 1A). Importantly, Notch receptors were present in both Ph-negative and Ph-like B-ALL samples (Fig. 1A).

Figure 1.

Notch receptors are expressed in B-ALL, and Notch activation results in apoptosis. A, Percentage of Notch1 to Notch4 receptor–positive cells of patients with B-ALL samples, and a representative cell line was determined by flow cytometry as the fraction of cells exceeding the intensity of the corresponding isotype control. Patients are represented by the same symbol for each receptor with PH-ve and PH-like subtypes discriminated by green and blue, respectively. Mean percentage for each receptor is represented by the horizontal bar. B, Patients with B-ALL samples (n = 3) were cultured on plate-bound human DLL1-Fc, DLL3-Fc, DLL4-Fc, Jagged1-Fc, Jagged2-Fc, and an IgG-Fc control. Viability was determined every 24 hours as the percentage of cells PI positive by flow cytometry. C, Cell lines representing T-ALL (CEM) and B-ALL (SB, JM1, Nalm6) and samples from Ph-like B-ALL patients (Pt.B1, Pt.B2) were cocultured on human bone marrow stromal HS5 cells expressing GFP (control) or DLL1. Viability was determined every 24 hours by Trypan blue (n = 3). D, Representative histograms of Annexin V positivity in the same cells cocultured with HS5 cells expressing DLL1 or control at 96 hours. E, RNA expression of Notch downstream genes HES1, DTX1, and HEY1 in patients with B-ALL samples (n = 3) cocultured for 24 hours on HS5 cells expressing GFP or DLL1, with and without a DLL1-blocking antibody, was assessed by qRT-PCR. F, A representative Western blot analysis from the same experiment depicting HES1(Abcam ab) at the protein level.

Figure 1.

Notch receptors are expressed in B-ALL, and Notch activation results in apoptosis. A, Percentage of Notch1 to Notch4 receptor–positive cells of patients with B-ALL samples, and a representative cell line was determined by flow cytometry as the fraction of cells exceeding the intensity of the corresponding isotype control. Patients are represented by the same symbol for each receptor with PH-ve and PH-like subtypes discriminated by green and blue, respectively. Mean percentage for each receptor is represented by the horizontal bar. B, Patients with B-ALL samples (n = 3) were cultured on plate-bound human DLL1-Fc, DLL3-Fc, DLL4-Fc, Jagged1-Fc, Jagged2-Fc, and an IgG-Fc control. Viability was determined every 24 hours as the percentage of cells PI positive by flow cytometry. C, Cell lines representing T-ALL (CEM) and B-ALL (SB, JM1, Nalm6) and samples from Ph-like B-ALL patients (Pt.B1, Pt.B2) were cocultured on human bone marrow stromal HS5 cells expressing GFP (control) or DLL1. Viability was determined every 24 hours by Trypan blue (n = 3). D, Representative histograms of Annexin V positivity in the same cells cocultured with HS5 cells expressing DLL1 or control at 96 hours. E, RNA expression of Notch downstream genes HES1, DTX1, and HEY1 in patients with B-ALL samples (n = 3) cocultured for 24 hours on HS5 cells expressing GFP or DLL1, with and without a DLL1-blocking antibody, was assessed by qRT-PCR. F, A representative Western blot analysis from the same experiment depicting HES1(Abcam ab) at the protein level.

Close modal

Activation of Notch signaling has antileukemic effects in B-ALL

Given the presence of all Notch receptors and the variety in receptor distribution, we next sought to determine whether cell death would be preferentially induced by individual Notch ligands. Plate-bound DLL1-Fc, Jagged1-Fc, or DLL4-Fc were all able to decrease viability in patient with B-ALL samples (Fig. 1B). In contrast, culture with DLL3-Fc or Jagged2-Fc had limited impact on cell viability. Notably, no ligand induced B-ALL cell growth (Fig. 1B). Although several Notch ligands could induce B-ALL cell death, we selected the most potent ligand, DLL1, as our model ligand. Annexin V positivity was observed in a dose-dependent manner when patient samples were cultured with increasing concentrations of DLL1-Fc (Supplementary Fig. S1A). Similarly, B-ALL cell lines and patient samples cocultured with human stromal cells overexpressing DLL1 (HS5-DLL1) exhibited decreased growth (Fig. 1C) and increased apoptosis (Fig. 1D) compared with those cultured with an HS5-GFP control. In line with previous work, this effect was not seen in a T-ALL cell line (Fig. 1C and D). In support of active Notch signaling, B-ALL cells cultured on HS5-DLL1 demonstrated a consistent increase in Notch downstream target genes HES1, HEY1, and DTX1 mRNA (Fig. 1E) and in HES1 protein (Fig. 1F) when compared with control cocultures. The addition of a DLL1-blocking antibody (xDLL1) to coculture mitigated the induction of HES1 protein expression (Fig. 1F), suggesting that the additional HES1 expression was due to the additional DLL1 ligand on these HS5 cells (HS5-DLL1). Induction of HES1 expression was seen across B-ALL cell lines and patient samples (Supplementary Fig. S1B). Together, these results illustrate that ligand-mediated Notch activation is capable of suppressing growth and survival of B-ALL.

Notch activation results in decreased PLK1 expression in a B-ALL–specific manner

Previous work showed that B-ALL cell death was induced by expression of the intracellular domain of any of the 4 Notch receptors (12). Here, we demonstrate that at least 3 recombinant Notch ligands induced significant B-ALL cell death (Fig. 1B). These observations suggest that B-ALL cells apoptose via a common Notch-mediated mechanism, which is potentially shared to different degrees by many Notch ligand–receptor pairs. Given the challenge of translating Notch-activating therapeutics, we sought to target the downstream mechanism of Notch activation in B-ALL cells. PLK1, a putatively oncogenic kinase, is stabilized by a negative regulator of Notch in melanoma (34). Hematologic malignancies, including ALL, reportedly express high levels of PLK1 (35, 36). We thus chose to examine whether the inverse was true—namely, if Notch activation was a negative regulator of PLK1 expression in B-ALL cells, ultimately leading to apoptosis.

Flow cytometry demonstrated that our B-ALL cell lines and patient samples did express PLK1 (Fig. 2A and C). Although expression was observed across B-ALL samples of various Ph-status, PLK1 was highest in Ph-like B-ALLs (Fig. 2B and D). Upon coculture with HS5-DLL1 cells, B-ALL cell lines and patient samples demonstrated marked reduction in the expression of PLK1 protein in the context of activation of Notch signaling, supported by the appearance of cleaved intracellular Notch2 (ICN2) and the common Notch target gene HES1 (Fig. 2E and G). Notably, Ph-like samples had the greatest reduction in PLK1 in response to DLL1-Notch activation (Fig. 2D). Expression of the intracellular domain of any of the 4 Notch receptors was capable of decreasing PLK1 expression (Fig. 2F), although ICN2 and ICN1 elicited the greatest reductions, and ICN4 only had a marginal decrease. These results are consistent with prior work demonstrating that expression of ICN1-4 could induce B-ALL cell death (12), and the activating potencies of Notch receptors, that is, ICN1 = ICN2 > ICN3 > ICN4.

Figure 2.

PLK1 is expressed in B-ALL and is suppressed by Notch activation. Intracellular PLK1 expression in cell lines (top) and patient samples (bottom) representing the 3 B-ALL subsets (A): PH−, PH-like, and PH+ was assessed by flow cytometry, and median fluorescence intensity (MFI; B) was summarized. Pale and dark shades represent cell lines and patient samples, respectively, within each subset. PH-like cells had significantly higher PLK1 MFI when compared with both PH− and PH+ cells (P < 0.01) using a one-way ANOVA with post hoc Tukey test. C, Intracellular PLK1 expression in the same cells was measured after 48 hours of coculture with HS5 cells expressing DLL1. D, Summary of the relative reduction of PLK1 MFI after coculture with HS5-DLL1 compared with baseline. PLK1 MFI reduction was significantly greater in PH-like when compared with PH− cells (P < 0.05) using a one-way ANOVA with post hoc Tukey test. E, T-ALL (CEM; a HES1-positive control) and B-ALL cell lines along with two representative Ph-like patients with B-ALL samples (Pt.B1, Pt.B2) were assessed by Western blot analysis for cleaved intracellular Notch 2 (ICN2), HES1(abcam ab), PLK1, and β-actin after coculture on HS5 cells expressing either GFP or DLL1 for 48 hours. F, Representative flow plots of PLK1 expression from the B-ALL cell line, SB, expressing intracellular Notch domains 1 to 4 (ICN1–ICN4) or a GFP control 48 hours postinduction. G, T-ALL (CEM, SupT1, Molt4, Jurkat) and B-ALL (SB, JM1, Nalm6, 697) cells were transduced with GFP control or HES1. HES1 (Origene ab) and PLK1 protein expression in transduced cells was determined by Western blot analysis.

Figure 2.

PLK1 is expressed in B-ALL and is suppressed by Notch activation. Intracellular PLK1 expression in cell lines (top) and patient samples (bottom) representing the 3 B-ALL subsets (A): PH−, PH-like, and PH+ was assessed by flow cytometry, and median fluorescence intensity (MFI; B) was summarized. Pale and dark shades represent cell lines and patient samples, respectively, within each subset. PH-like cells had significantly higher PLK1 MFI when compared with both PH− and PH+ cells (P < 0.01) using a one-way ANOVA with post hoc Tukey test. C, Intracellular PLK1 expression in the same cells was measured after 48 hours of coculture with HS5 cells expressing DLL1. D, Summary of the relative reduction of PLK1 MFI after coculture with HS5-DLL1 compared with baseline. PLK1 MFI reduction was significantly greater in PH-like when compared with PH− cells (P < 0.05) using a one-way ANOVA with post hoc Tukey test. E, T-ALL (CEM; a HES1-positive control) and B-ALL cell lines along with two representative Ph-like patients with B-ALL samples (Pt.B1, Pt.B2) were assessed by Western blot analysis for cleaved intracellular Notch 2 (ICN2), HES1(abcam ab), PLK1, and β-actin after coculture on HS5 cells expressing either GFP or DLL1 for 48 hours. F, Representative flow plots of PLK1 expression from the B-ALL cell line, SB, expressing intracellular Notch domains 1 to 4 (ICN1–ICN4) or a GFP control 48 hours postinduction. G, T-ALL (CEM, SupT1, Molt4, Jurkat) and B-ALL (SB, JM1, Nalm6, 697) cells were transduced with GFP control or HES1. HES1 (Origene ab) and PLK1 protein expression in transduced cells was determined by Western blot analysis.

Close modal

Importantly, overexpression of HES1, the most commonly reported Notch downstream target gene, was sufficient to decrease PLK1 protein expression in B-ALL cell lines, but not in T-ALL cell lines (Fig. 2G), suggesting that the Notch/HES-mediated downregulation of PLK1 occurs in a B-ALL–specific manner.

PLK1 is a survival kinase in B-ALL that mediates p53 suppression

Given these observations that Notch activation downregulates PLK1 expression, we sought to determine whether direct downregulation of PLK1 expression was sufficient to inhibit growth and survival in patient B-ALL cells. B-ALL with shPLK1 exhibited a significant decrease in viability, but this effect was not seen in T-ALL (Fig. 3A and B). This reduction in cell viability with partial PLK1 downregulation suggests the importance of PLK1 in the survival of B-ALL cells.

Figure 3.

PLK1 inhibition promotes B-ALL–specific cell death and is associated with p53 accumulation. A, Primary cells from B-ALL (left) and T-ALL (right) patients (each n = 3) were transduced with doxycycline-inducible PLK1 or scrambled shRNAs and cultured in low-dose doxycycline (2 μg/mL). Cell viability was determined on days 1 and 4 by Trypan blue staining. B, The PLK1-shRNA knockdown was confirmed both at the RNA and protein level by qRT-PCR and Western blot analysis, respectively. PLK1 RNA was significantly reduced in both patients with B-ALL samples (P < 0.05, n = 3) by two-sample t tests. C, Representative flow cytometry data summarizing the differences in intracellular p53 expression from a patient with T-ALL and 5 patients with B-ALL samples transduced with the PLK1 or scrambled shRNA after 48 hours.

Figure 3.

PLK1 inhibition promotes B-ALL–specific cell death and is associated with p53 accumulation. A, Primary cells from B-ALL (left) and T-ALL (right) patients (each n = 3) were transduced with doxycycline-inducible PLK1 or scrambled shRNAs and cultured in low-dose doxycycline (2 μg/mL). Cell viability was determined on days 1 and 4 by Trypan blue staining. B, The PLK1-shRNA knockdown was confirmed both at the RNA and protein level by qRT-PCR and Western blot analysis, respectively. PLK1 RNA was significantly reduced in both patients with B-ALL samples (P < 0.05, n = 3) by two-sample t tests. C, Representative flow cytometry data summarizing the differences in intracellular p53 expression from a patient with T-ALL and 5 patients with B-ALL samples transduced with the PLK1 or scrambled shRNA after 48 hours.

Close modal

In other malignancies, PLK1 is proposed to exert oncogenic functions by suppressing the proapoptotic function of p53 (20, 37). In contrast, p53 can repress transcription of PLK1 in response to DNA damage (38). To explore this inverse relationship between PLK1 and p53, we evaluated p53 expression in B-ALL samples following PLK1 knockdown. p53 expression was significantly increased in shPLK1 cells compared with control, and this effect was not seen in T-ALL cells (Fig. 3C). Together, this demonstrates that PLK1 is required for survival in B-ALL, and may exert its effects in part through suppression of p53.

Notch-induced loss of PLK1 stabilizes p53 through MDM2 hypophosphorylation

PLK1 has been shown to modulate p53 stability and function via phosphorylation of MDM2 at Ser260, resulting in p53 degradation (21). To evaluate whether these PLK1–p53 relationships are maintained in B-ALL, we tested the effect of activated Notch signaling on pMDM2Ser260 and p53 levels in B-ALL cell lines and patient with B-ALL samples. Notch ligand-mediated activation dramatically decreased pMDM2Ser260 without altering total MDM2 levels, and markedly increased p53 protein levels (Fig. 4A). Importantly, inhibition of Notch receptor cleavage with DAPT, a γ-secretase inhibitor that acts as a nonspecific inhibitor of Notch activation, could abrogate the majority of the p53 accumulation (Fig. 4B) and attenuate HES1 induction in the context of Notch ligand exposure (Fig. 4C), supporting the hypothesis that this mechanism is driven by canonical Notch signaling.

Figure 4.

PLK1 inhibition prevents MDM2 phosphorylation and increases p53 accumulation in B-ALL. A, Ph-like patient with B-ALL samples (Pt.B1, Pt.B2) and B-ALL cell line, SB, were cocultured on HS5 cells expressing either the GFP-control or DLL1. MDM2 (phospho and total), p53, PLK1, HES1, and β-actin protein expression was quantified by Western blot analysis. B, Intracellular p53 was measured in primary B-ALL patient cells (Pt.B1) treated with soluble DLL1, γ-secretase (Notch) inhibitor DAPT (200 nmol/L), or both for 48 hours by flow cytometry. C, Similarly, HES1 was quantified by qRT-PCR in the same primary B-ALL cells treated with DLL1, DAPT, or both, relative to untreated controls. Only the DLL1-treated cells had a significant increase in HES1 RNA expression (P < 0.05) by one-way ANOVA with Dunnett post hoc test. D, A representative Ph-like patient with B-ALL sample was cultured on either control (Fc) or DLL1 (DLL1Fc) plate-bound ligand for 48 hours. Cell lysates were immunoprecipitated with IgG control, MDM2, or p53, and the membrane was probed with pMDM2 and p53. E and F, Similarly, intracellular pMDM2 and p53 were measured by flow cytometry for 2 patients with B-ALL samples after 48 hours of culture on control (Fc) or DLL1 (DLL1Fc) plate-bound ligand. Intracellular p53 expression was significantly increased in both patient samples (P < 0.05 and P < 0.01) cultured on DLL1Fc when compared with their corresponding control cultures.

Figure 4.

PLK1 inhibition prevents MDM2 phosphorylation and increases p53 accumulation in B-ALL. A, Ph-like patient with B-ALL samples (Pt.B1, Pt.B2) and B-ALL cell line, SB, were cocultured on HS5 cells expressing either the GFP-control or DLL1. MDM2 (phospho and total), p53, PLK1, HES1, and β-actin protein expression was quantified by Western blot analysis. B, Intracellular p53 was measured in primary B-ALL patient cells (Pt.B1) treated with soluble DLL1, γ-secretase (Notch) inhibitor DAPT (200 nmol/L), or both for 48 hours by flow cytometry. C, Similarly, HES1 was quantified by qRT-PCR in the same primary B-ALL cells treated with DLL1, DAPT, or both, relative to untreated controls. Only the DLL1-treated cells had a significant increase in HES1 RNA expression (P < 0.05) by one-way ANOVA with Dunnett post hoc test. D, A representative Ph-like patient with B-ALL sample was cultured on either control (Fc) or DLL1 (DLL1Fc) plate-bound ligand for 48 hours. Cell lysates were immunoprecipitated with IgG control, MDM2, or p53, and the membrane was probed with pMDM2 and p53. E and F, Similarly, intracellular pMDM2 and p53 were measured by flow cytometry for 2 patients with B-ALL samples after 48 hours of culture on control (Fc) or DLL1 (DLL1Fc) plate-bound ligand. Intracellular p53 expression was significantly increased in both patient samples (P < 0.05 and P < 0.01) cultured on DLL1Fc when compared with their corresponding control cultures.

Close modal

Adding further to this mechanism, coimmunoprecipitation assays in a B-ALL patient sample demonstrated loss of endogenous MDM2–p53 interaction following Notch activation (Fig. 4D). Likewise, flow cytometry revealed a decrease in pMDM2Ser260 and a corresponding increase in p53 staining intensity in these patient with B-ALL samples (Fig. 4E and F). Together, these results demonstrate that Notch-mediated PLK1 downregulation leads to a significant reduction of pMDM2Ser260 and subsequent p53 stabilization. This regulation of the MDM2:p53 interaction through PLK1-mediated phosphorylation of MDM2 at Ser260 is a novel mechanism in B-ALL and provides insight into Notch-mediated effects in B-ALL while also highlighting PLK1 as a potential therapeutic target in B-ALL.

Notch-HES1 signaling promotes CHFR-mediated ubiquitination of PLK1

To further understand the mechanism of Notch/HES-mediated downregulation of PLK1, we examined PLK1 mRNA expression following exposure to Notch ligand and found no significant differences (Fig. 5A), suggesting that Notch-mediated regulation of PLK1 protein levels occurs posttranslationally. Because PLK1 is known to be ubiquitinated and subsequently degraded by the proteasome (39, 40), we tested whether PLK1 protein was ubiquitinated following Notch activation. Following Notch activation, PLK1 was ubiquitinated in B-ALL cells, with ubiquitination seen at 24 and 48 hours (Fig. 5B and C). PLK1 was ubiquitinated in the same cells following HES1 overexpression (Fig. 5D), confirming the role for Notch signaling in the ubiquitination of PLK1.

Figure 5.

Notch-mediated PLK1 ubiquitination involves PARylation of CHFR1 (checkpoint ubiquitin E3 ligase) in B-ALL. A, PLK1 mRNA expression was determined by qRT-PCR after coculture with HS5-DLL1 or HS5-GFP for 48 hours (n = 3). B, B-ALL cell lines were treated with MG132 4 hours prior to various time points (12, 24, or 48 hours) of coculture with HS5-GFP or HS5-DLL1. Cell lysates were subjected to immunoprecipitation with ubiquitin and probed for PLK1. C, Similarly, after 48 hours of coculture, cells were treated with MG132 for 4 hours and harvested. Cell lysates were subjected to immunoprecipitation with PLK1, and blots were probed with PLK1 and ubiquitin. D, The same panel of B-ALL cells was transduced with retrovirus-mediated HES1 for 48 hours to induce ectopic expression of the gene. The cells were treated with MG132 and the cell lysates subjected to immunoprecipitation with ubiquitin and probed for PLK1. E, Primary B-ALL cells (Pt. B1) were either transduced with HES1 retrovirus, transfected with CHFR siRNA (siCHFR), or both for 24 hours; these cells and untreated controls were probed for CHFR and PLK1 expression. F, Cell lysates from B-ALL cell lines coculture with HS5-GFP or HS5-DLL1 were immunoprecipitated with CHFR and probed for PLK1. G, T-ALL cells (CEM) and B-ALL cells (SB, JM1, Nalm6) were either cocultured on control or DLL1-expressing HS5 cells and subjected to CHFR depletion via siRNA for 4 days. Viability was determined by Trypan blue. H, Cell lysates from E were immunoprecipitated with p53, and blots were probed with pMDM2(ser260) and p53. I, B-ALL cells were cocultured on control or DLL1-expressing HS5 cells for 48 hours, and cell lysates were immunoprecipitated with CHFR and resolved on 4% to 15% non-denaturing gradient gels; the membrane was probed for polyADP ribosylation (PAR). J, Cells from patients with Ph-like B-ALL (Pt.B1, Pt.B2) were cocultured on control or DLL1-expressing HS5 cells and treated for 48 hours with the PARP inhibitor, 3ABA. Viability was determined by Trypan blue (*, P < 0.05; **, P < 0.01).

Figure 5.

Notch-mediated PLK1 ubiquitination involves PARylation of CHFR1 (checkpoint ubiquitin E3 ligase) in B-ALL. A, PLK1 mRNA expression was determined by qRT-PCR after coculture with HS5-DLL1 or HS5-GFP for 48 hours (n = 3). B, B-ALL cell lines were treated with MG132 4 hours prior to various time points (12, 24, or 48 hours) of coculture with HS5-GFP or HS5-DLL1. Cell lysates were subjected to immunoprecipitation with ubiquitin and probed for PLK1. C, Similarly, after 48 hours of coculture, cells were treated with MG132 for 4 hours and harvested. Cell lysates were subjected to immunoprecipitation with PLK1, and blots were probed with PLK1 and ubiquitin. D, The same panel of B-ALL cells was transduced with retrovirus-mediated HES1 for 48 hours to induce ectopic expression of the gene. The cells were treated with MG132 and the cell lysates subjected to immunoprecipitation with ubiquitin and probed for PLK1. E, Primary B-ALL cells (Pt. B1) were either transduced with HES1 retrovirus, transfected with CHFR siRNA (siCHFR), or both for 24 hours; these cells and untreated controls were probed for CHFR and PLK1 expression. F, Cell lysates from B-ALL cell lines coculture with HS5-GFP or HS5-DLL1 were immunoprecipitated with CHFR and probed for PLK1. G, T-ALL cells (CEM) and B-ALL cells (SB, JM1, Nalm6) were either cocultured on control or DLL1-expressing HS5 cells and subjected to CHFR depletion via siRNA for 4 days. Viability was determined by Trypan blue. H, Cell lysates from E were immunoprecipitated with p53, and blots were probed with pMDM2(ser260) and p53. I, B-ALL cells were cocultured on control or DLL1-expressing HS5 cells for 48 hours, and cell lysates were immunoprecipitated with CHFR and resolved on 4% to 15% non-denaturing gradient gels; the membrane was probed for polyADP ribosylation (PAR). J, Cells from patients with Ph-like B-ALL (Pt.B1, Pt.B2) were cocultured on control or DLL1-expressing HS5 cells and treated for 48 hours with the PARP inhibitor, 3ABA. Viability was determined by Trypan blue (*, P < 0.05; **, P < 0.01).

Close modal

Because the E3 ligase CHFR has been reported to target PLK1 for ubiquitination (40), we sought to investigate whether CHFR was important for PLK1 ubiquitination in B-ALL. Indeed, knockdown of CHFR impaired HES1-mediated loss of PLK1 expression (Fig. 5E), linking HES1 to CHFR and PLK1. We then confirmed an interaction between PLK1 and CHFR via immunoprecipitation of endogenous proteins in B-ALL cell lines (Fig. 5F). To determine whether CHFR was necessary for the Notch-mediated growth inhibition in B-ALL, we partially knocked down CHFR via siRNA and observed partial rescue of Notch ligand-mediated reductions in B-ALL cell counts, but not in a T-ALL cell line (Fig. 5G), suggesting an important role for CHFR in this mechanism. In line with the notion that this cell death is due, at least in part, to p53 stabilization, we sought to understand the role of CHFR in p53 stabilization in B-ALL. Indeed, CHFR knockdown prevented the loss of pMDM2Ser260 and the stabilization of p53 in patient with B-ALL cells (Fig. 5H).

Notch-mediated PLK1 degradation involves PARylation of CHFR

Our previous work demonstrated that HES1 activates PARP1, resulting in global poly-ADP-ribosylation (PARylation) in B-ALL (13). Following exposure to DLL1, CHFR was found to be PARylated in B-ALL cells (Fig. 5I). Addition of a PARP1 inhibitor (3ABA) rescued the majority of Notch ligand-mediated growth inhibition in B-ALL samples (Fig. 5J), underscoring the importance of CHFR and PARP activity in this mechanism of Notch-mediated growth inhibition.

The experiments described above reveal a novel mechanism of Notch/HES-mediated PARP/CHFR-mediated PLK1 loss and subsequent dysregulation of MDM2/p53, leading to growth arrest and apoptosis in B-ALL cells. However, as Notch ligands/agonists are a challenge to translate into a clinical therapeutic, the remainder of the manuscript focuses on the use of PLK1 inhibitors as a clinically available method to mimic Notch-mediated apoptosis in B-ALL.

Pharmacologic PLK1 inhibition mimics Notch-induced effects in B-ALL

The PLK1 inhibitor BI2536 dramatically decreased cell growth of B-ALL cell lines and patient samples, but had little effect on a T-ALL cell line (Fig. 6A). Consistent with the Notch/HES-mediated mechanism revealed above, exposure to BI2536 resulted in a decrease in pMDM2Ser260 and subsequent p53 stabilization in B-ALL, but not T-ALL (Fig. 6B and C). These results were further corroborated by Western blot analysis (Fig. 6D). These data confirm the importance of this PLK1/MDM2/p53 mechanism in B-ALL and demonstrate the therapeutic potential of PLK1 inhibition in this disease.

Figure 6.

Pharmacologic inhibition of PLK1 shows antileukemia activity in Ph-like B-ALL. A, Cells representing T-ALL (CEM) or B-ALL (SB, JM1, Nalm6 [N6]) and primary samples from patients with Ph-like B-ALL were treated with a vehicle control or PLK1 inhibitor, BI2536 (100 nmol/L), for 1 week. Cells were counted by Trypan blue daily and the counts normalized the seed number. B and C, Intracellular p53 (B) and pMDM2 (ser260; C) for all cells in A were determined by flow cytometry at 48 hours. D, Similarly, cell lysates were collected at the same time point and probed for pMDM2 (ser260), MDM2, p53, and β-actin. E, NSG-SGM3 mice engrafted with cells from a patient with Ph-like B-ALL (Pt.B1) were treated, beginning 3 weeks after initial tail vein injection, with vehicle (control), PLK1 inhibitor BI2536 (15 mg/kg), or BI6727 (volasertib; 15 mg/kg) by gavage twice per week for 2 weeks (n = 10 mice per group) and continuously monitored for 2 additional weeks. The leukemia burden was measured weekly in the peripheral blood by hCD45 staining. F, Mice were euthanized 4 weeks after beginning treatment, and the leukemia burden was measured in the bone marrow and spleen by hCD45 staining. G, Intracellular pMDM2 and p53 levels in the bone marrow of the treated mice were quantified by flow-based analysis. H, SB B-ALL was treated with various combinations of dexamethasone (DEX) or vincristine (Vi) and PLK1 inhibitor BI2536 (BI) for 3 days (n = 3 per treatment group). Cell viability was determined by Alamar blue (*, P < 0.05; **, P < 0.01).

Figure 6.

Pharmacologic inhibition of PLK1 shows antileukemia activity in Ph-like B-ALL. A, Cells representing T-ALL (CEM) or B-ALL (SB, JM1, Nalm6 [N6]) and primary samples from patients with Ph-like B-ALL were treated with a vehicle control or PLK1 inhibitor, BI2536 (100 nmol/L), for 1 week. Cells were counted by Trypan blue daily and the counts normalized the seed number. B and C, Intracellular p53 (B) and pMDM2 (ser260; C) for all cells in A were determined by flow cytometry at 48 hours. D, Similarly, cell lysates were collected at the same time point and probed for pMDM2 (ser260), MDM2, p53, and β-actin. E, NSG-SGM3 mice engrafted with cells from a patient with Ph-like B-ALL (Pt.B1) were treated, beginning 3 weeks after initial tail vein injection, with vehicle (control), PLK1 inhibitor BI2536 (15 mg/kg), or BI6727 (volasertib; 15 mg/kg) by gavage twice per week for 2 weeks (n = 10 mice per group) and continuously monitored for 2 additional weeks. The leukemia burden was measured weekly in the peripheral blood by hCD45 staining. F, Mice were euthanized 4 weeks after beginning treatment, and the leukemia burden was measured in the bone marrow and spleen by hCD45 staining. G, Intracellular pMDM2 and p53 levels in the bone marrow of the treated mice were quantified by flow-based analysis. H, SB B-ALL was treated with various combinations of dexamethasone (DEX) or vincristine (Vi) and PLK1 inhibitor BI2536 (BI) for 3 days (n = 3 per treatment group). Cell viability was determined by Alamar blue (*, P < 0.05; **, P < 0.01).

Close modal

Therapeutic PLK1 inhibition demonstrates antileukemia activity in B-ALL PDX models

To evaluate whether PLK1 inhibition reduces leukemia burden in vivo, we treated a Ph-like B-ALL PDX mouse model with the PLK1 inhibitors BI2536 or BI6727 (volasertib). Strikingly, mice that received either PLK1 inhibitor had leukemic burden reductions of 70% in blood (Fig. 6E), 90% in bone marrow, and 95% in spleen compared with mice receiving vehicle (Fig. 6F). Analysis of bone marrow cells isolated from mice receiving BI2636 or BI6727 revealed a decrease in pMDM2Ser260 with concomitant p53 stabilization compared with vehicle-treated mice (Fig. 6G). In addition to increased p53, mice that received BI6727 also had increased expression of the proapoptotic protein BAX in leukemia blasts, suggesting a further downstream mechanism of apoptosis (S2a-f). These data support the existence of a PLK1–MDM2–p53 mechanism in B-ALL and demonstrate the potential therapeutic efficacy of direct PLK1 inhibition in B-ALL.

PLK1 inhibition enhances efficacy of standard chemotherapeutics in B-ALL

Although PLK1 inhibition appears to have marked antileukemic effects in B-ALL, single-agent therapies are only rarely utilized clinically. Thus, we evaluated whether addition of a PLK1 inhibitor would confer any additional benefit to standardly used chemotherapeutic agents. When combined with dexamethasone or vincristine, a low dose of BI2536 (5-fold lower than previous in vitro experiments) significantly reduced cell viability compared with any of the 3 agents alone (Fig. 6H). These suggest that the antileukemia effects of dexamethasone and vincristine can be potentiated by PLK1 inhibition.

Levels of PARP1 and MDM2 transcripts are higher in patients with B-ALL samples than in patients with T-ALL samples

Our data suggest an important role for Notch-mediated PLK1 inhibition, occurring via PARylation of CHFR, hypophosphorylation of MDM2Ser260, stabilization of p53, and upregulation of proapoptotic proteins such as BAX. To evaluate whether this mechanism could be reasonably generalized across a larger subset of B-ALL, we analyzed data from 172 bone marrow samples from B-ALL cases (representing all major biologic subtypes) and 34 T-ALL cases compared with 9 leukemia-free donors. Supplementary Figure S3 displays a heat map of the relative mRNA expression of the genes involved in this mechanism. Of the Notch receptors, transcript levels of NOTCH1 and NOTCH2 were highest, consistent with their relatively abundant surface expression (Fig. 1A). HES1 levels were elevated in T-ALL, but were uniformly low in B-ALL, consistent with the constitutive Notch activation seen in many T-ALL cases, with low HES1 protein (Fig. 1E and F) and with our prior findings of a lack of constitutive Notch signaling in B-ALL (12, 13). CHFR and PLK1 were expressed at similar levels in B-ALL and T-ALL, although these transcript expression array data do not reflect the posttranslational regulation described here. Likewise, TP53 levels appear similar between B-ALL and T-ALL. Importantly, both PARP1 and MDM2 transcripts were significantly higher in B-ALL than T-ALL, potentially revealing why B-ALL is sensitive to the PLK1–MDM2–p53 mechanism described here.

This study demonstrates that Notch ligand-mediated Notch activation induces B-ALL–specific growth inhibition and apoptosis, in part through PLK1 reduction. Our data demonstrate that PLK1 is ubiquitinated by CHFR, which is PARylated by PARP1 in response to Notch activation via HES1 expression. The decrease in PLK1 results in MDM2 hypophosphorylation, p53 stabilization, and upregulation of proapoptotic proteins (BAX).

It appears that multiple Notch ligands and receptors may contribute to this mechanism. DLL1 was used as the model ligand in these studies, although multiple Notch ligands were able to induce apoptosis (Fig. 1B), suggesting that this mechanism is not specific to a single ligand. Although patient samples had variable distribution of Notch receptors (Fig. 1A; Supplementary Fig. S3), each sample tested exhibited PLK1 downregulation and ultimately cell death in response to Notch activation. Likewise, expression of ICN1-4 or HES1 phenocopied Notch-ligand–mediated PLK1 downregulation (Fig. 2F and G), suggesting that any Notch receptor can induce this mechanism through HES gene upregulation. Finally, DAPT abrogated the effects of Notch activation on PLK1 downregulation in B-ALL (Fig. 4C and D) suggesting that this mechanism relies on cleavage of the Notch receptors and canonical Notch signaling. Together, these observations allude to a general Notch-HES pathway effect, that is not exclusive to one ligand or receptor. Deciphering relative contributions of each Notch receptor or ligand in different patient samples may be helpful in further understanding the physiologic effects of Notch signaling in B-ALL. However, by therapeutically targeting the downstream mechanism via direct PLK inhibition, the contribution of Notch pathway becomes less relevant for the clinic.

In this study, PLK1 inhibitor dramatically potentiated the effects of both vincristine and dexamethasone (Fig. 6H). These may suggest that addition of a PLK1-targeted agent may reduce the exposure to chemotherapy necessary to achieve clinical response. To this end, PLK1 inhibitors, including volasertib (BI6727), have shown acceptable toxicity profiles and have had modest clinical response in combination with other agents for advanced solid tumors and acute myeloid leukemia (41–43). Although PLK1 inhibition has not been used clinically in B-ALL, accumulating evidence supports a role for PLK1 in B-cell oncogenesis. In B-cell lymphoma, overexpression of PLK1 has been described as an independent prognostic factor (44). A recent publication has demonstrated a pivotal role for PLK1 in double-hit diffuse large B-cell lymphoma, and proposed combination therapy of PLK1 inhibition and venetoclax, a BCL2 inhibitor, in this disease (45). This aligns nicely with our current study, in which PLK1 inhibition upregulated p53 and the proapoptotic BAX protein, ultimately resulting in B-ALL growth arrest and apoptosis. Future studies using these combinations in B-ALL would certainly be of use given the widespread use and clinical efficacy of venetoclax (46, 47).

In this study, we utilized samples with wild-type p53. The majority of pediatric B-ALL presents with wild-type p53, although relapse is associated with a higher incidence of TP53 mutations (48). In adult B-ALL, p53 is mutated in ∼15% of newly diagnosed cases (49–51). More frequently, other mechanisms of p53 inactivation—such as deregulation of MDM2 or ARF—occur (52, 53). This is also evident in our analysis of patient samples, where MDM2 levels were elevated (Supplementary Fig. S3). Hypophosphorylation of MDM2 by Notch-HES1–mediated PLK1 reduction appears to act as a means to reactivate wild-type p53, which results in upregulation of proapoptotic proteins, such as BAX (Supplementary Fig. S2). Future studies evaluating whether this mechanism remains intact when p53 is mutated in B-ALL would be worth exploring.

Previously, we showed that HES1 interacts with and activates PARP1 in B-ALL, resulting in nuclear translocation of apoptosis inducing factor (AIF), caspase cleavage, and apoptosis of B-ALL cells (13). In this study, we expand on these findings by further relating PARP1 to PARylation and activation of CHFR, which is required for the ubiquitination and subsequent degradation of PLK1 in B-ALL. These mechanisms are likely to work in concert with one another, which may explain why knockdown of CHFR only partially rescues DLL1-mediated reductions in B-ALL cell counts (Fig. 5G). Therefore, a therapeutic means to selectively activate Notch in B-ALL cells may ultimately be more beneficial than PLK1 inhibition in isolation. The antileukemic activity of Notch signaling in B-ALL resides in striking contrast to the oncogenic Notch activation seen in various malignancies, including T-ALL (54, 55). This reasonably causes hesitation regarding the notion of developing Notch-activating therapeutics to treat B-ALL. Should such a venture be evaluated, ensuring specificity to B-lineage cells via conjugation of Notch ligand to an antibody raised against an antigen such as CD19, CD20, or CD22 may be useful.

Notch-activation induced B-ALL apoptosis appears to be a phenomenon observed across B-ALL subtypes (Fig. 2). Intriguingly, this effect is particularly pronounced in Ph-like samples, representing a high-risk phenotype (Fig. 2D). Likewise, Ph-like cell lines and patient samples had the highest PLK1 expression (Fig. 2C), which may brand this subtype as particularly amenable to PLK1 inhibition. These compelling findings require further investigation.

In summary, this study demonstrated a role for active Notch-HES signaling in downregulating PLK1 in a PARP1/CHFR-dependent manner. This led to hypophosphorylation of MDM2, stabilization of p53, and upregulation of the proapoptotic protein BAX, ultimately resulting in B-ALL cell death. PLK1 inhibitors had marked proapoptotic effects in vitro and in PDX models of B-ALL. Together, these data strongly suggest that Notch activators or PLK1 inhibitors are viable therapeutic approaches in B-ALL that warrant clinical investigation.

M. Konopleva has ownership interest (including stock, patents, etc.) in Reata Pharmaceutical, and is a consultant/advisory board member for Abbvie, Genentech, F. Hoffman LaRoche, Cellectis, and Stemline Therapeutics. C.G. Mullighan reports of receiving Loxo Oncoloy, Pfizer, and Abbvie; has received speakers bureau honoraria from Amgen, Pfizer, and Illumina; and is a consultant/advisory board member for St. Justine CHU. P.A. Zweidler-McKay is a Senior Medical Director at and has ownership interest (including stock, patents, etc.) in ImmunoGen. No potential conflicts of interest were disclosed by the other authors.

Conception and design: S. Kannan, M.J.L. Aitken, S.M. Herbrich, J. Chandra, P.A. Zweidler-McKay

Development of methodology: S. Kannan, L.S. Golfman, J.K. Burks, P.A. Zweidler-McKay

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Kannan, D.H. Mak, J.K. Burks, M. Konopleva, C.G. Mullighan, P.A. Zweidler-McKay

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Kannan, M.J.L. Aitken, S.M. Herbrich, M.G. Hall, J.K. Burks, G. Song, C.G. Mullighan, J. Chandra, P.A. Zweidler-McKay

Writing, review, and/or revision of the manuscript: S. Kannan, M.J.L. Aitken, S.M. Herbrich, J.K. Burks, C.G. Mullighan, J. Chandra, P.A. Zweidler-McKay

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Kannan, S.M. Herbrich

Study supervision: S. Kannan, J. Chandra, P.A. Zweidler-McKay

Other (assisted with blood and tissue sampling and flow analysis.): L.S. Golfman

Other (secured funding for the study.): J. Chandra

This work was supported by the NCI (R01CA138816, to P.A. Zweidler-McKay) and by a research grant from Alex's Lemonade Stand (ALSF ID# 4497, to J. Chandra). Additional funding support was provided by Cancer Prevention & Research Institute of Texas (CPRIT: RP150006, to M. Konopleva) and by a Laboratory Incentive Fund from the Division of Pediatrics (to S. Kannan), and additional resources from Richard Gorlick, MD. Thanks to Dr. Michael Roth (Division of Pediatrics) and the Department of Scientific Publications (MD Anderson Cancer Center) for reviewing the manuscript. The authors are grateful to Drs. Michelle Barton, Dean of the University of Texas Graduate School of Biomedical Sciences, and Faye M. Johnson, Associate Professor of Thoracic/Head and Neck Medical Oncology at MD Anderson Cancer Center (Houston, TX), for helpful discussion about the manuscript and providing MDM2 and p53 reagents. The authors also are thankful to the Cellular Imaging Core, Department of Leukemia, for their valuable support in analyzing the CyTOF samples with the support of the NIH/NCI through the MD Anderson Cancer Center Support Grant (CCSG) under award number P30CA016672.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Kato
M
,
Manabe
A
. 
Treatment and biology of pediatric acute lymphoblastic leukemia
.
Pediatr Int
2018
;
60
:
4
12
.
2.
Jabbour
E
,
O'Brien
S
,
Konopleva
M
,
Kantarjian
H
. 
New insights into the pathophysiology and therapy of adult acute lymphoblastic leukemia
.
Cancer
2015
;
121
:
2517
28
.
3.
Mody
R
,
Li
S
,
Dover
DC
,
Sallan
S
,
Leisenring
W
,
Oeffinger
KC
, et al
Twenty-five-year follow-up among survivors of childhood acute lymphoblastic leukemia: a report from the Childhood Cancer Survivor Study
.
Blood
2008
;
111
:
5515
23
.
4.
Vrooman
LM
,
Silverman
LB
. 
Treatment of childhood acute lymphoblastic leukemia: prognostic factors and clinical advances
.
Curr Hematol Malig Rep
2016
;
11
:
385
94
.
5.
Mullighan
CG
,
Collins-Underwood
JR
,
Phillips
LA
,
Loudin
MG
,
Liu
W
,
Zhang
J
, et al
Rearrangement of CRLF2 in B-progenitor- and Down syndrome-associated acute lymphoblastic leukemia
.
Nat Genet
2009
;
41
:
1243
6
.
6.
Roberts
KG
,
Morin
RD
,
Zhang
J
,
Hirst
M
,
Zhao
Y
,
Su
X
, et al
Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia
.
Cancer Cell
2012
;
22
:
153
66
.
7.
Jain
N
,
Roberts
KG
,
Jabbour
E
,
Patel
K
,
Eterovic
AK
,
Chen
K
, et al
Ph-like acute lymphoblastic leukemia: a high-risk subtype in adults
.
Blood
2017
;
129
:
572
81
.
8.
Ness
KK
,
Armenian
SH
,
Kadan-Lottick
N
,
Gurney
JG
. 
Adverse effects of treatment in childhood acute lymphoblastic leukemia: general overview and implications for long-term cardiac health
.
Expert Rev Hematol
2011
;
4
:
185
97
.
9.
Maude
SL
,
Laetsch
TW
,
Buechner
J
,
Rives
S
,
Boyer
M
,
Bittencourt
H
, et al
Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia
.
N Engl J Med
2018
;
378
:
439
48
.
10.
Kantarjian
H
,
Stein
A
,
Gokbuget
N
,
Fielding
AK
,
Schuh
AC
,
Ribera
JM
, et al
Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia
.
N Engl J Med
2017
;
376
:
836
47
.
11.
Kantarjian
HM
,
DeAngelo
DJ
,
Stelljes
M
,
Martinelli
G
,
Liedtke
M
,
Stock
W
, et al
Inotuzumab ozogamicin versus standard therapy for acute lymphoblastic leukemia
.
N Engl J Med
2016
;
375
:
740
53
.
12.
Zweidler-McKay
PA
,
He
Y
,
Xu
L
,
Rodriguez
CG
,
Karnell
FG
,
Carpenter
AC
, et al
Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies
.
Blood
2005
;
106
:
3898
906
.
13.
Kannan
S
,
Fang
W
,
Song
G
,
Mullighan
CG
,
Hammitt
R
,
McMurray
J
, et al
Notch/HES1-mediated PARP1 activation: a cell type-specific mechanism for tumor suppression
.
Blood
2011
;
117
:
2891
900
.
14.
Maillard
I
,
Fang
T
,
Pear
WS
. 
Regulation of lymphoid development, differentiation, and function by the Notch pathway
.
Annu Rev Immunol
2005
;
23
:
945
74
.
15.
Hori
K
,
Sen
A
,
Artavanis-Tsakonas
S
. 
Notch signaling at a glance
.
J Cell Sci
2013
;
126
(
Pt 10
):
2135
40
.
16.
Kageyama
R
,
Ohtsuka
T
. 
The Notch-HES pathway in mammalian neural development
.
Cell Res
1999
;
9
:
179
88
.
17.
Weng
AP
,
Ferrando
AA
,
Lee
W
,
Morris
JP 4th
,
Silverman
LB
,
Sanchez-Irizarry
C
, et al
Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia
.
Science
2004
;
306
:
269
71
.
18.
Aster
JC
,
Pear
WS
,
Blacklow
SC
. 
Notch signaling in leukemia
.
Annu Rev Pathol
2008
;
3
:
587
613
.
19.
Tamagnone
L
,
Zacchigna
S
,
Rehman
M
. 
Taming the Notch transcriptional regulator for cancer therapy
.
Molecules
2018
;
23
:
431
.
20.
Ando
K
,
Ozaki
T
,
Yamamoto
H
,
Furuya
K
,
Hosoda
M
,
Hayashi
S
, et al
Polo-like kinase 1 (Plk1) inhibits p53 function by physical interaction and phosphorylation
.
J Biol Chem
2004
;
279
:
25549
61
.
21.
Dias
SS
,
Hogan
C
,
Ochocka
AM
,
Meek
DW
. 
Polo-like kinase-1 phosphorylates MDM2 at Ser260 and stimulates MDM2-mediated p53 turnover
.
FEBS Lett
2009
;
583
:
3543
8
.
22.
Liu
Z
,
Sun
Q
,
Wang
X
. 
PLK1, a potential target for cancer therapy
.
Transl Oncol
2017
;
10
:
22
32
.
23.
Liu
X
,
Erikson
RL
. 
Polo-like kinase (Plk)1 depletion induces apoptosis in cancer cells
.
Proc Natl Acad Sci U S A
2003
;
100
:
5789
94
.
24.
Pui
JC
,
Allman
D
,
Xu
L
,
DeRocco
S
,
Karnell
FG
,
Bakkour
S
, et al
Notch1 expression in early lymphopoiesis influences B versus T lineage determination
.
Immunity
1999
;
11
:
299
308
.
25.
Privette
LM
,
Gonzalez
ME
,
Ding
L
,
Kleer
CG
,
Petty
EM
. 
Altered expression of the early mitotic checkpoint protein, CHFR, in breast cancers: implications for tumor suppression
.
Cancer Res
2007
;
67
:
6064
74
.
26.
Kannan
S
,
Sutphin
RM
,
Hall
MG
,
Golfman
LS
,
Fang
W
,
Nolo
RM
, et al
Notch activation inhibits AML growth and survival: a potential therapeutic approach
.
J Exp Med
2013
;
210
:
321
37
.
27.
Purnell
MR
,
Whish
WJ
. 
Novel inhibitors of poly(ADP-ribose) synthetase
.
Biochem J
1980
;
185
:
775
7
.
28.
Rudolph
D
,
Steegmaier
M
,
Hoffmann
M
,
Grauert
M
,
Baum
A
,
Quant
J
, et al
BI 6727, a Polo-like kinase inhibitor with improved pharmacokinetic profile and broad antitumor activity
.
Clin Cancer Res
2009
;
15
:
3094
102
.
29.
Steegmaier
M
,
Hoffmann
M
,
Baum
A
,
Lenart
P
,
Petronczki
M
,
Krssak
M
, et al
BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo
.
Curr Biol
2007
;
17
:
316
22
.
30.
Krutzik
PO
,
Trejo
A
,
Schulz
KR
,
Nolan
GP
. 
Phospho flow cytometry methods for the analysis of kinase signaling in cell lines and primary human blood samples
.
Methods Mol Biol
2011
;
699
:
179
202
.
31.
Nolo
R
,
Herbrich
S
,
Rao
A
,
Zweidler-McKay
P
,
Kannan
S
,
Gopalakrishnan
V
. 
Targeting P-selectin blocks neuroblastoma growth
.
Oncotarget
2017
;
8
:
86657
70
.
32.
Herbrich
SM
,
Kannan
S
,
Nolo
RM
,
Hornbaker
M
,
Chandra
J
,
Zweidler-McKay
PA
. 
Characterization of TRKA signaling in acute myeloid leukemia
.
Oncotarget
2018
;
9
:
30092
105
.
33.
Qiu
P
,
Simonds
EF
,
Bendall
SC
,
Gibbs
KD
 Jr
,
Bruggner
RV
,
Linderman
MD
, et al
Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE
.
Nat Biotechnol
2011
;
29
:
886
91
.
34.
Schmit
TL
,
Nihal
M
,
Ndiaye
M
,
Setaluri
V
,
Spiegelman
VS
,
Ahmad
N
. 
Numb regulates stability and localization of the mitotic kinase PLK1 and is required for transit through mitosis
.
Cancer Res
2012
;
72
:
3864
72
.
35.
Ikezoe
T
,
Yang
J
,
Nishioka
C
,
Takezaki
Y
,
Tasaka
T
,
Togitani
TK
, et al
A novel treatment strategy targeting polo-like kinase 1 in hematological malignancies
.
Leukemia
2009
;
23
:
1564
76
.
36.
Hartsink-Segers
SA
,
Exalto
C
,
Allen
M
,
Williamson
D
,
Clifford
SC
,
Horstmann
M
, et al
Inhibiting Polo-like kinase 1 causes growth reduction and apoptosis in pediatric acute lymphoblastic leukemia cells
.
Haematologica
2013
;
98
:
1539
46
.
37.
Bussey
KJ
,
Bapat
A
,
Linnehan
C
,
Wandoloski
M
,
Dastrup
E
,
Rogers
E
, et al
Targeting polo-like kinase 1, a regulator of p53, in the treatment of adrenocortical carcinoma
.
Clin Transl Med
2016
;
5
:
1
.
38.
McKenzie
L
,
King
S
,
Marcar
L
,
Nicol
S
,
Dias
SS
,
Schumm
K
, et al
p53-dependent repression of polo-like kinase-1 (PLK1)
.
Cell Cycle
2010
;
9
:
4200
12
.
39.
Kim
JS
,
Park
YY
,
Park
SY
,
Cho
H
,
Kang
D
,
Cho
H
. 
The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells
.
J Biol Chem
2011
;
286
:
30615
23
.
40.
Kang
D
,
Chen
J
,
Wong
J
,
Fang
G
. 
The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition
.
J Cell Biol
2002
;
156
:
249
59
.
41.
Schoffski
P
,
Awada
A
,
Dumez
H
,
Gil
T
,
Bartholomeus
S
,
Wolter
P
, et al
A phase I, dose-escalation study of the novel Polo-like kinase inhibitor volasertib (BI 6727) in patients with advanced solid tumours
.
Eur J Cancer
2012
;
48
:
179
86
.
42.
Gjertsen
BT
,
Schoffski
P
. 
Discovery and development of the Polo-like kinase inhibitor volasertib in cancer therapy
.
Leukemia
2015
;
29
:
11
9
.
43.
Dohner
H
,
Lubbert
M
,
Fiedler
W
,
Fouillard
L
,
Haaland
A
,
Brandwein
JM
, et al
Randomized, phase 2 trial of low-dose cytarabine with or without volasertib in AML patients not suitable for induction therapy
.
Blood
2014
;
124
:
1426
33
.
44.
Liu
L
,
Zhang
M
,
Zou
P
. 
Expression of PLK1 and survivin in diffuse large B-cell lymphoma
.
Leuk Lymphoma
2007
;
48
:
2179
83
.
45.
Ren
Y
,
Bi
C
,
Zhao
X
,
Lwin
T
,
Wang
C
,
Yuan
J
, et al
PLK1 stabilizes a MYC-dependent kinase network in aggressive B cell lymphomas
.
J Clin Invest
2018
;
128
:
5517
30
.
46.
Roberts
AW
,
Davids
MS
,
Pagel
JM
,
Kahl
BS
,
Puvvada
SD
,
Gerecitano
JF
, et al
Targeting BCL2 with venetoclax in relapsed chronic lymphocytic leukemia
.
N Engl J Med
2016
;
374
:
311
22
.
47.
Konopleva
M
,
Pollyea
DA
,
Potluri
J
,
Chyla
B
,
Hogdal
L
,
Busman
T
, et al
Efficacy and biological correlates of response in a phase II study of venetoclax monotherapy in patients with acute myelogenous leukemia
.
Cancer Discov
2016
;
6
:
1106
17
.
48.
Hof
J
,
Krentz
S
,
van Schewick
C
,
Korner
G
,
Shalapour
S
,
Rhein
P
, et al
Mutations and deletions of the TP53 gene predict nonresponse to treatment and poor outcome in first relapse of childhood acute lymphoblastic leukemia
.
J Clin Oncol
2011
;
29
:
3185
93
.
49.
Chiaretti
S
,
Brugnoletti
F
,
Tavolaro
S
,
Bonina
S
,
Paoloni
F
,
Marinelli
M
, et al
TP53 mutations are frequent in adult acute lymphoblastic leukemia cases negative for recurrent fusion genes and correlate with poor response to induction therapy
.
Haematologica
2013
;
98
:
e59
61
.
50.
Stengel
A
,
Schnittger
S
,
Weissmann
S
,
Kuznia
S
,
Kern
W
,
Kohlmann
A
, et al
TP53 mutations occur in 15.7% of ALL and are associated with MYC-rearrangement, low hypodiploidy, and a poor prognosis
.
Blood
2014
;
124
:
251
8
.
51.
Kanagal-Shamanna
R
,
Jain
P
,
Takahashi
K
,
Short
NJ
,
Tang
G
,
Issa
GC
, et al
TP53 mutation does not confer a poor outcome in adult patients with acute lymphoblastic leukemia who are treated with frontline hyper-CVAD-based regimens
.
Cancer
2017
;
123
:
3717
24
.
52.
Mullighan
CG
,
Phillips
LA
,
Su
X
,
Ma
J
,
Miller
CB
,
Shurtleff
SA
, et al
Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia
.
Science
2008
;
322
:
1377
80
.
53.
Iacobucci
I
,
Ferrari
A
,
Lonetti
A
,
Papayannidis
C
,
Paoloni
F
,
Trino
S
, et al
CDKN2A/B alterations impair prognosis in adult BCR-ABL1-positive acute lymphoblastic leukemia patients
.
Clin Cancer Res
2011
;
17
:
7413
23
.
54.
Ferrando
AA
,
Herblot
S
,
Palomero
T
,
Hansen
M
,
Hoang
T
,
Fox
EA
, et al
Biallelic transcriptional activation of oncogenic transcription factors in T-cell acute lymphoblastic leukemia
.
Blood
2004
;
103
:
1909
11
.
55.
Demarest
RM
,
Ratti
F
,
Capobianco
AJ
. 
It's T-ALL about Notch
.
Oncogene
2008
;
27
:
5082
91
.