There is limited understanding of how signaling pathways are altered by oncogenic fusion transcription factors that drive leukemogenesis. To address this, we interrogated activated signaling pathways in a comparative analysis of mouse and human leukemias expressing the fusion protein E2A-PBX1, which is present in 5%–7% of pediatric and 50% of pre-B-cell receptor (preBCR+) acute lymphocytic leukemia (ALL). In this study, we describe remodeling of signaling networks by E2A-PBX1 in pre-B-ALL, which results in hyperactivation of the key oncogenic effector enzyme PLCγ2. Depletion of PLCγ2 reduced proliferation of mouse and human ALLs, including E2A-PBX1 leukemias, and increased disease-free survival after secondary transplantation. Mechanistically, E2A-PBX1 bound promoter regulatory regions and activated the transcription of its key target genes ZAP70, SYK, and LCK, which encode kinases upstream of PLCγ2. Depletion of the respective upstream kinases decreased cell proliferation and phosphorylated levels of PLCγ2 (pPLCγ2). Pairwise silencing of ZAP70, SYK, or LCK showed additive effects on cell growth inhibition, providing a rationale for combination therapy with inhibitors of these kinases. Accordingly, inhibitors such as the SRC family kinase (SFK) inhibitor dasatinib reduced pPLCγ2 and inhibited proliferation of human and mouse preBCR+/E2A-PBX1+ leukemias in vitro and in vivo. Furthermore, combining small-molecule inhibition of SYK, LCK, and SFK showed synergistic interactions and preclinical efficacy in the same setting. Our results show how the oncogenic fusion protein E2A-PBX1 perturbs signaling pathways upstream of PLCγ2 and renders leukemias amenable to targeted therapeutic inhibition. Cancer Res; 76(23); 6937–49. ©2016 AACR.
Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and one of the main causes of cancer-related death in children and young adults. ALL is a heterogeneous disease and can be subclassified by karyotype, cell type, and immunophenotype. One distinctive immunophenotype is characterized by expression and tonic functional activity of the pre-B-cell receptor (preBCR). Recent studies indicate that half of preBCR+ ALLs harbor the chromosomal translocation t(1:19), which codes for the chimeric transcription factor E2A-PBX1(1, 2). Although E2A-PBX1+ ALLs are associated with an intermediate risk, about 20% of younger patients and 70% of adult patients with E2A-PBX1+ ALL die 5 years after diagnosis, with relapse being one of the main causes of death (3, 4). Although the treatment and prognosis of patients with pediatric ALL have improved during the last decades, there is a clinical need for more effective/selective and less toxic therapies, particularly in this distinctive ALL subset and in preBCR+ ALL in general.
To address this, we employed a comparative approach based on human and mouse E2A-PBX1 leukemias using biochemical and genetic functional assays. We identified activated signaling pathways that lead to higher phosphorylation levels of PLCγ2, which plays a crucial role in cell survival and proliferation. The chimeric transcription factor E2A-PBX1 transcriptionally regulates genes that code for key kinases upstream of PLCγ2, resulting in signaling network remodeling in pre-B-ALLs. Small-molecule inhibitors targeting specifically these pathways show promising preclinical efficacy in vitro and in vivo and should be considered in future clinical trials to treat E2A-PBX1+/preBCR+ ALLs.
Materials and Methods
Conditional E2A-PBX1 transgenic mice were reported previously (5). Transgenic CD19.Cre (The Jackson Laboratory; ref. 6), Mb1.Cre (provided by Dr. David Allman, University of Pennsylvania, Philadelphia, PA, and by Dr. Michael Reth, University of Freiburg, Freiburg im Breisgau, Germany; ref. 7), and Mx1.Cre (The Jackson Laboratory; ref. 8) mice were intercrossed to generate E2A-PBX1/CD19.Cre, E2A-PBX1/Mb1.Cre, and E2A-PBX1/Mx1.Cre mice, respectively, on a C57BL/6 background. Leukemia cells derived from E2A-PBX1/CD19.Cre and E2A-PBX1/Mx1.Cre mice, which were preBCR+ as seen by cytoplasmic μ chain, were used for in vitro and in vivo experiments. Leukemia cells derived from E2A-PBX1/Mb1.Cre mice, which were preBCR−, were used for in vitro experiments. Disease-free survival was defined as the time from transplantation to the time when the mice showed signs of illness, including general lymphadenopathy, lethargy, weight loss, and shivering. Moribund mice were euthanized.
Human leukemia cell lines 697, RCH-ACV, Kasumi-2, SEM, RS4;11, REH, and HAL-01 (obtained from DSMZ) were cultured in RPMI1640 medium supplemented with 10 % FBS, 100 U/mL penicillin/streptomycin, and 0.29 mg/mL l-glutamine. SUP-B15 cells (obtained from ATCC) were cultured in RPMI1640 medium supplemented with 20% FBS. E2A-PBX1+ cell lines (697, RCH-ACV, Kasumi-2) were authenticated using Western blot analysis for E2A-PBX1 fusion protein expression. E2A-PBX1− cell lines (SEM, RS4;11, REH, HAL-01) were obtained from DSMZ in 2013 and not further authenticated. PreBCR status was assessed by flow cytometry for surface VPREB. Primary human ALL samples were obtained from the Tissue Bank of the Department of Pediatrics, Stanford University (Stanford, CA).
Flow cytometry and FACS
Bone marrow cells from transgenic mice were prepared as described previously (5). Flow cytometry was performed in LSR Fortessa (BD Biosciences) and FACS sorting in FACS Aria (BD Biosciences) using FACS DIVA software (BD Biosciences) and FlowJo (Treestar) for analysis. Antibodies used for flow cytometry analysis and FACS sorting are listed in Supplementary Table S1. Lineage-negative (Lin−) cells were detected with a cocktail of antibodies including anti-CD3, CD4, CD8, Mac1, Gr1, NK1.1, and Ter119.
Murine and human leukemia cells were preincubated in DMEM high-glucose medium (Thermo Scientific) containing 10 % FBS for 1 hour at 37 °C, and then treated for 30 minutes at 37°C using following small-molecule inhibitors: dasatinib (LC Laboratories), saracatinib (Selleckchem), bosutinib (Selleckchem), P505-15 (Selleckchem), R406 (Selleckchem), R778 (fostamatinib, Selleckchem), RK24466 (Cayman Chemical), LCKi-II (Thermo Fisher Scientific), A-770041 (Axon Medchem), ibrutinib (PCI-32765, Selleckchem), buparlisib (Selleckchem), or trametinib (LC Laboratories).
For intracellular staining, cells were fixed with 1.5% formaldehyde for 10 minutes at room temperature, permeabilized with 100% ice-cold methanol for 20 minutes on ice, and washed twice with staining buffer as described previously (9, 10), and stained with conjugated antibodies to intracellular phospho-proteins (Supplementary Table S2). Data were analyzed using Flowjo software.
Bone marrow transplantation assays and in vivo drug treatment
Leukemia cells were transduced with lentiviral vectors containing shRNA for luciferase (control) or indicated genes and mCherry fluorescence reporter. mCherry+ cells were sorted 7 days after transduction. Secondary bone marrow transplantation assay using 1,000 mouse preBCR+/E2A-PBX1+ leukemia cells per recipient was described elsewhere (5). For in vivo treatment, mice were treated daily, intraperitoneally, for 21 days, starting the day after transplantation with vehicle (30% PEG1500, 1% Tween 80, 2.5 % DMSO dissolved in PBS), 10 mg/kg bodyweight (b.w.) P505-15 (Selleckchem; ref. 11) or 5 mg/kg b.w. A-770041 (Axon Medchem; ref. 12).
Colony-forming assays and in vitro drug treatment
Mouse leukemia cells (1,000/well) were cultured in methylcellulose medium (M3234, StemCell Technologies) supplemented with 10 ng/mL IL7 (Miltenyi Biotec). Human leukemia cells (2,000/well) were cultured in methylcellulose medium (H4230, StemCell Technologies). Colonies from mouse leukemias were counted after 7 days, and from human leukemia cell lines after 14 days. Leukemia cells were cultured in the presence of dasatinib, saracatinib, bosutinib, P505-15, R406, R778, RK24466, LCKi-II, A-770041, ibrutinib, buparlisib, and trametinib at the concentrations indicated.
RNA was isolated using the RNeasy Mini Kit (Qiagen) and cDNA was synthesized using Superscript Reverse Transcriptase III kit (Life Technologies) following the manufacturer's recommendations. Relative expression was quantified using an ABI 7900HT Thermocycler with TaqMan Master Mix (Applied Biosystems) at an annealing temperature of 60°C using TaqMan Gene Expression Assays from Life Technologies (Supplementary Table S3). All signals were quantified using the ΔΔCt method and normalized to ΔΔCt values of the Actb gene expression levels.
Gene expression microarray
Bone marrow cells from mouse E2A-PBX1 leukemic mice and B-cell progenitors (lin−CD19+CD43+) from wild-type and preleukemic mice (GFP− or GFP+) were used. Healthy preleukemic mice were defined as 3-month-old transgenic mice without any sign of disease. RNA used for microarray analysis was prepared using an RNeasy Mini Kit (Qiagen). Gene 1.0 ST arrays (Affymetrix) were used according to the manufacturer's instructions. Normalizations of CEL file data, comparison between groups, and generation of heatmaps were performed using software Nexus Expression (Biodiscover). The Gene Expression Omnibus (http://ncbi.nlm.nih.gov/geo) accession number for the microarray raw data reported in this article is GSE81010.
shRNA knockdown vectors and transduction
For knockdown studies, the shRNAs were designed using a commercial web tool (Invitrogen). Individual shRNA sequences (Supplementary Table S4) were cloned into the BstXI site of the p309 lentiviral vector (13, 14), for stable transduction in mouse E2A-PBX1 leukemia cells and human ALL cell lines.
Lentivirus generation and transduction of mouse and human cells are described elsewhere (15). The sorted mCherry+ cells were cultured for 7 days for colony-forming assay or used in secondary bone marrow transplantation experiments (1,000 cells each secondary recipient).
Chromatin immunoprecipitation and genomic qPCR
Chromatin immunoprecipitation (ChIP) assays were performed as described elsewhere (16). Briefly, cells were harvested and fixed with 1% fresh formaldehyde and the immunocomplexes were precipitated using GFP-Trap or control magnetic agarose beads (gtma-20 and bmab-20, respectively, ChromoTek Inc.). Quantitative real-time PCR (qRT-PCR) was performed on the precipitated DNA using primers flanking the ZAP70, SYK, LCK, TBP, and NCAPD2 genes. Previously published ChIP-seq peaks for E2A-PBX1 (1) within regulatory regions described in immortalized cell lines analyzed in the ENCODE project (17) were chosen for validation as well as control sequences in promoter or intergenic regions. The relative values to input were determined using genomic qPCR with SYBR Green as fluorescence dye. Primers are listed in Supplementary Table S5.
Western blot analysis
Western blot analysis for protein quantification was performed using a modified RIPA lysis buffer as described previously (5). Following antibodies were used for immunodetection: rat anti-E2A (clone 826927; R&D Systems), rabbit anti-PLCγ2 (#3872S, Cell Signaling Technology), mouse anti-ZAP70 (clone 1E7.2, Merck Millipore), mouse anti-SYK (clone SYK-01, Biolegend), rabbit anti-LCK (#2752S, Cell Signaling Technology), rabbit anti-histone H3 (#Ab1791, Abcam) mouse anti-tubulin (GenScript), or rabbit anti-Gapdh (#G9545, Sigma-Aldrich) antibodies. Secondary antibodies mouse anti-rat ((#HAF005, R&D Systems), rabbit anti-mouse (#816729, Life Technologies), and goat anti-rabbit (#G21234, Life Technologies) coupled to HRP were used. Quantification by densitometry was performed using ImageJ software (NIH, Bethesda, MD; ref. 18).
Statistical differences between two groups were analyzed by two-sided Mann–Whitney U test or by Student t test. Dose–response curves were compared using the extra sum of squares F test. The Bliss independence model (19) was used to evaluate synergy between drug combinations. A P value below 0.05 was considered statistically significant. Statistical differences from Kaplan–Meier curves were analyzed by log-rank (Mantel–Cox) test. Statistical analysis and graphs were performed using GraphPad prism software (GraphPad Inc.).
All experiments on mice were performed with the approval of and in accordance with Stanford's Administrative Panel on Laboratory Animal Care (APLAC, Protocol 9839).
Oncogenic role for hyperphosphorylated pPLCγ2 in pre-B-ALL
Various signaling pathways were interrogated in engineered mouse strains that conditionally activate and express E2A-PBX1 and develop ALL (5). Phospho-flow analysis revealed hyperphosphorylation of PLCγ2 in B-cell progenitors from E2A-PBX1 bone marrow leukemia cells compared with healthy wild-type mice [about 7-fold difference of median fluorescence intensity (MFI)], consistent with constitutive hyperactivation of upstream signaling pathways. (Fig. 1A and B).
The role of PLCγ2 has been extensively characterized as a key enzyme in B-cell receptor signaling in mature B cells (20); however, its role in B-cell progenitors and pre-B-cell leukemogenesis is less well known. To study the functional role of PLCγ2 in mouse and human E2A-PBX1 leukemias, an shRNA knockdown approach was employed using lentiviral transduction (13, 14).Efficient shRNA-mediated knockdown of PLCγ2 (Fig. 1C) reduced clonogenicity in mouse E2A-PBX1+ leukemia cells, independently of the presence or absence of the preBCR (Fig. 1D). Although E2A-PBX1+/preBCR+ leukemias have increased clonogenicity compared with E2A-PBX1+/preBCR− (P = 0.029), no differences in pPLCγ2 were observed consistent with shRNA knockdown experiments (data not shown). In secondary bone marrow transplantation experiments, recipients of PLCγ2-depleted mouse leukemia cells showed an extended disease-free survival compared with controls (Fig. 1E). Similarly, efficient depletion of PLCγ2 by shRNA in human ALL cell lines (Fig. 1F) correlated with reduced clonogenicity (Fig. 1G and H), suggesting a pathogenic role of hyperactivated PLCγ2 not only in E2A-PBX1+ but also in B-ALL leukemogenesis in general.
Identification of E2A-PBX1 target genes upstream of PLCγ2
Transcriptional and bioinformatics analyses were employed to identify candidate signaling proteins that may serve key roles in hyperactivation of PLCγ2 and leukemia pathogenesis. Gene ontology analysis of E2A-PBX1 target genes in human ALLs (1) revealed several biological processes that lead to the activation of PLCγ2 including leukocyte and lymphocyte activation pathways (Fig. 2A). From the gene lists associated with these gene ontology terms, we identified three candidate kinases that are upstream of PLCγ2: the cytoplasmic kinases ZAP70 (zeta chain associated protein) and SYK (spleen tyrosine kinase), and the SRC family kinase LCK (lymphocyte-specific protein tyrosine kinase).
The genes for all three of these kinases showed consistent enrichment for proteins reactive with E2A and PBX1 antibodies in publicly available ChIP-seq datasets (Supplementary Fig. S1; ref. 1). Using ChIP-PCR assays, we confirmed that E2A-PBX1 localizes to sites identified by ChIP-seq in the vicinity of these genes (Fig. 2B) within regions that display chromatin features of gene-regulatory elements defined by DNaseI hypersensitivity peaks in immortalized cell lines from the ENCODE project (17) (Supplementary Fig. S1). Furthermore, shRNA-mediated E2A-PBX1-depletion resulted in decreased levels of SYK, LCK, and ZAP70 (Fig. 2C and D), indicating that E2A-PBX1 binds and activates their transcription in pre-B-ALL.
Consistent upregulation of ZAP70, SYK, and LCK in E2A-PBX1 transformation
Gene expression patterns in mouse leukemias and preleukemic transgenic B-cell progenitors, as well as in human primary ALLs (21) induced by the E2A-PBX1 oncogene were compared with the transcriptional profile of normal B-cell progenitors. Intersection of the microarray datasets revealed ZAP70 as the only gene, other than E2A-PBX1, that is upregulated in all E2A-PBX1–expressing cells [Fig. 3A and B, mouse E2A-PBX1 leukemias vs. normal B-cell progenitors (P < 0.001); preleukemic B-cell progenitors GFP+ vs. GFP− (P < 0.05); human E2A-PBX1 vs. normal B-cell progenitors (P < 0.01)]. These data suggest that ZAP70 is one of the earliest and most consistently upregulated genes during E2A-PBX1 leukemogenesis. Increased ZAP70 expression does not reflect merely differentiation stage after comparison with closely related (lin−CD19+CD43+) progenitor B cells (Fig. 3C). Increased ZAP70 protein was also observed in human E2A-PBX1 primary ALL cells and cell lines (Fig. 3C) consistent with previous observations (22).
Expression of ZAP70, SYK, and LCK genes was assessed in human primary ALLs using a publicly available dataset (Fig. 3D and Supplementary Fig. S2A) and the Oncomine database (Supplementary Fig. S2B; ref. 23). E2A-PBX1+ primary ALLs showed higher expression levels of all three kinases compared with non-E2A-PBX1 primary ALLs in both cohorts of patients, reinforcing further our conclusion that E2A-PBX1 activates transcription of these kinase genes. Their higher level expression in human and mouse E2A-PBX1–expressing cells strongly suggests a pathogenic role.
Oncogenic roles of the E2A-PBX1 target genes ZAP70, SYK, and LCK in pre-B-ALLs
The functional roles of ZAP70, SYK, and LCK in E2A-PBX1 leukemias were studied using an shRNA knockdown approach. Efficient depletion of each kinase in human (Fig. 4A) and mouse (Supplementary Fig. S3A) E2A-PBX1 leukemia cells correlated with decreased clonogenicity (Fig. 4B and Supplementary Fig. S3B). Sublethally irradiated mice transplanted with kinase-depleted mouse leukemia cells showed extended disease-free survival, demonstrating an important in vivo role during leukemogenesis (Supplementary Fig. S3C). Interestingly, E2A-PBX1+ cells were more susceptible to ZAP70 depletion as shown by reduced cell proliferation compared with non-E2A-PBX1 cells (Fig. 4C). Phospho-flow analysis of shRNA-transduced cells showed that depletion of ZAP70, LCK, or SYK resulted in decreased pPLCγ2 in E2A-PBX1+ cells (Fig. 4D) and in E2A-PBX1− cells (data not shown). These results confirmed that the kinases are upstream of PLCγ2 and may serve as possible pharmacologic targets to decrease cellular levels of pPLCγ2 in pre-B-ALL.
Pharmacologic inhibition of PLCγ2 upstream signaling pathways in E2A-PBX1 leukemias
Several small-molecule inhibitors were evaluated for their effects on PLCγ2 upstream pathways in E2A-PBX1 leukemia cells. SRC family kinase (SFK) inhibitors including dasatinib as well as inhibitors of SYK and LCK were effective in reducing pPLCγ2 in ALL cells, consistent with shRNA knockdown studies (Fig. 5A). The effect of SFK, SYK, and LCK, inhibitors was specific to pPLCγ2 decrease as compared with stable pSTAT5 levels using the same conditions (data not shown). Preclinical efficacy was tested in vitro comparing IC50 concentrations in colony-forming assays of mouse preBCR+ and preBCR− leukemias (Fig. 5B). PreBCR+ leukemias were more sensitive to the SFK inhibitor dasatinib, as described previously (5), as well as to SYK inhibitors P505-15 (Fig. 5C and D) and R406 (data not shown) and LCK inhibitors A-770041 (Fig. 5C and D) and RK2446 (data not shown). The preclinical efficacy of P505-15 and A-770041 was tested in vivo after secondary bone marrow transplantation of preBCR+ leukemias (11, 12). As expected, in vivo treatment with SYK and LCK inhibitors prolonged disease-free survival of mice (Fig. 5E). These data identified novel compounds, which inhibit specifically the proliferation of preBCR+/E2A-PBX1+ leukemias compared with their genetically similar preBCR−/E2A-PBX1+ leukemias and show promising preclinical efficacy.
Genetic interactions among E2A-PBX1 target genes reveal effective combination therapies
The partial decrease in pPLCγ2 after pharmacologic inhibition and shRNA-mediated knockdown targeting individually ZAP70, SYK, or LCK, and the modest effects on cell proliferation suggest that compensatory effects might be occurring in these pathways. Therefore, genetic interactions between E2A-PBX1 target genes in human ALL cells were studied using vectors coexpressing shRNAs with mCherry or GFP as fluorescence markers (Supplementary Fig. S4A). Cotransduced cells were monitored using fluorescence microscopy or flow cytometry (Supplementary Fig. S4B and S4C). Cosuppression of ZAP70 and LCK in RCH-ACV and 697 cells showed a statistically significant additive inhibition of cell growth (Fig. 6A). A trend for additive inhibition was observed following cosuppression of ZAP70 and SYK (Supplementary Fig. S4D) as well as LCK and SYK (Supplementary Fig. S4E), suggesting that genetic inactivation of a specific kinase might increase the efficacy of inhibitors targeting compensatory kinases.
Thus, competition assays of shRNA-transduced cells were performed (Supplementary Fig. S5A). Cocultured mCherry+ (shRNA for gene of interest) and GFP+ (control shRNA) transduced cells were monitored over time by flow cytometry in the presence of small-molecule inhibitors (Fig. 6B). Compared with control cells, cells depleted for ZAP70 showed a decreased competitive fitness, that was further reduced by dasatinib (SFKi), R406 (SYKi), or A-770041 (LCKi; Fig. 6C). Similar results were observed for cells depleted of SYK (Supplementary Fig. S5B) or LCK (Supplementary Fig. S5C), confirming genetic interactions between E2A-PBX1 target genes. Of note, SYK-depleted human cells showed markedly impaired cell proliferation consistent with reduced leukemogenesis of SYK-depleted mouse leukemias in secondary bone marrow transplantation (Supplementary Fig. S3C). These results provide a rationale for combination therapy to block the hyperactivated PLCγ2 signaling pathway at different levels in preBCR+ leukemias.
Preclinical efficacy of combination treatment with small molecules in preBCR+ E2A-PBX1+ leukemias
Finally, the ability of combination targeted therapy to block signaling pathways upstream of PLCγ2 was tested in human and mouse ALL cells. Human cell lines RCH-ACV (preBCR+/E2A-PBX1+) and SEM (preBCR−/E2A-PBX1−) were treated with A-770041 (LCKi) and either P505-15 or R406 (SYKi). Combination therapy inhibited synergistically the clonogenicity of RCH-ACV cells using the Bliss independence model (P < 0.05, Fig. 7A and B and Supplementary Fig. S6A; ref. 19). However, the addition of A-770041 (LCKi) to the SYK inhibitors did not affect the colony-forming capacity of SEM cells (Fig. 7B; data not shown), suggesting that the inhibitory effect of the combination therapy is specific for preBCR+/E2A-PBX1+ cells. We also tested whether SYK inhibitors might increase the sensitivity of human preBCR+/E2A-PBX1+ leukemias to dasatinib. Indeed, combination therapies with P505-15 (SYKi) and dasatinib (SFKi) inhibited synergistically clonogenicity of RCH-ACV compared with SEM cells (P <0.001, Fig. 7B).
To elucidate whether the enhanced effect of combination treatment is due to the presence of the preBCR, mouse preBCR+/E2A-PBX1+ and preBCR−/E2A-PBX1+ leukemias that arose on similar genetic backgrounds were assessed in vitro in drug titration experiments. Mouse preBCR+/E2A-PBX1+ leukemias were more sensitive to the combination therapy of A-770041 (LCKi) with either SYK inhibitor P505-15 (Fig. 7C) or R406 (Supplementary Fig. S6B). Similarly, cell growth inhibition by dasatinib (SFKi) was enhanced by P505-15 (Fig. 7D), A-770041, or R406 (Supplementary Fig. S6C) in preBCR+/E2A-PBX1+ but not in preBCR−/E2A-PBX1+ leukemias. The combination therapy of SYK inhibitor P505-15 with A-770041 (LCKi, P < 0.05) exhibited synergy as did the cotreatment with P505-15 and dasatinib (SFKi, P < 0.001).
Taken together, these data reveal novel genetic interactions of E2A-PBX1 target genes that form the rationale for combination therapies, which show promising in vitro and in vivo preclinical efficacy.
Aberrant activation of signaling pathways has been linked to leukemogenesis; however, little is known about cell signaling perturbations induced by fusion transcription factors. Using a cross-species comparative approach of human and mouse leukemias from novel engineered transgenic mice, we describe here signaling network remodeling by the chimeric fusion protein E2A-PBX1 in pre-B-ALL, which results in hyperactivation of PLCγ2. E2A-PBX1 binds regulatory elements and activates the transcription of its target genes ZAP70, SYK, and LCK, upstream of PLCγ2, which are essential for cell survival and proliferation. Consequently, several kinase inhibitors targeting signaling pathways upstream PLCγ2 display promising preclinical efficacy in preBCR+/E2A-PBX1+ leukemias (Fig. 7E).
A direct link of E2A-PBX1 with deregulated signaling pathways upstream of PLCγ2 is supported by multiple lines of evidence: E2A-PBX1 binds genomic regions of ZAP70, SYK, and LCK genes, respectively, that display chromatin features of gene regulatory elements; depletion of E2A-PBX1 by shRNA reduces the expression of these kinases; E2A-PBX1 expression in mouse and human primary ALLs as well as in human ALL cell lines positively correlates with elevated ZAP70, SYK, and LCK expression; and E2A-PBX1+ ALL cells are strongly dependent on expression of these kinases as demonstrated by knockdown and pharmacologic inhibition. As their expression was not absolutely restricted to E2A-PBX1+ ALLs, alternative mechanisms may drive their oncogenic roles in other ALL subtypes, and variable dependence on the kinases among individual E2A-PBX1+ ALLs likely reflects pathway redundancies, sample heterogeneity, or acquisition of secondary mutations affecting transcription or epigenetic factors involved in E2A-PBX1 target gene regulation.
ZAP70, SYK, and LCK have been previously implicated in hematologic malignancies other than B-cell precursor ALL. Expression of ZAP70 is associated with elevated downstream signaling (24, 25) and is an adverse prognostic marker in B-chronic lymphocytic leukemia (B-CLL; refs. 26, 27). LCK overexpression results from chromosomal translocation in T-cell leukemias (28–30). SYK overexpression has been described in peripheral T-cell lymphomas (31) as well as in chromosomal translocations of peripheral T-cell lymphomas and myelodysplastic syndromes (32–34).Our studies describe a novel mechanism of kinase overexpression by a chimeric fusion protein in B-cell precursor ALL. Using a comparative approach, we revealed the functional consequences as well as the in vitro and in vivo dependencies on overexpressed kinases. Hence, our findings significantly extend the spectrum of hematologic malignancies in which ZAP70, SYK, and LCK play important roles to enhance oncogenic signaling in malignant lymphoid cells.
Our pharmacologic screen identified inhibitors of SFKs, SYK, and LCK as compounds that inhibit activated signaling pathways upstream of pPLCγ2 in E2A-PBX1+ leukemia cells. Hence, dasatinib, SYK inhibitors, and LCK inhibitors specifically inhibit cell proliferation of preBCR+/E2A-PBX1+ leukemias. Dasatinib is a tyrosine kinase inhibitor (TKI) that is approved by the FDA for treatment of BCR-ABL+ CML and ALL (35). Recent preclinical studies have suggested that dasatinib may also be a candidate therapy for additional ALL subtypes including BCR-ABL–like ALL (36) and preBCR+ ALL, which includes E2A-PBX1+ pre-B-ALLs (1, 5, 37, 38).The SYK inhibitor R406/R778 (fostamatinib) is FDA-approved for patients with immune thrombocytopenic purpura and P505-15 is currently in phase II clinical trial for rheumatoid arthritis. SYK inhibition also showed preclinical efficacy in preBCR+ xenografts in previous studies (1, 2). Our data confirm and extend these previous observations, and for the first time demonstrate LCK inhibitors as small molecules with promising preclinical efficacy in preBCR+/E2A-PBX1+ ALL. In addition, we observed enhanced efficacy in combination therapies employing dasatinib, SYK, and LCK inhibitors, which might be relevant to prevent or ameliorate drug resistance in future clinical applications. Although LCK inhibitors have only been used as a research tool, the translation of the findings regarding dasatinib and SYK inhibitors for the treatment of preBCR+ ALL patients might be accelerated in future clinical trials.
Our genetic studies of individual kinase knockdowns showed a modest decrease in cell growth inhibition (Fig. 4B), which is enhanced by double shRNA knockdown (Fig. 6A). shRNA-mediated knockdown of individual kinases decreases pPLCγ2 (Fig. 4D). However, double shRNA knockdown does not further reduce pPLCγ2 levels (data not shown), suggesting some redundancy of PLCγ2 upstream kinases. Small molecules show a much more robust effect on cell proliferation and decrease of pPLCγ2 (Fig. 5A and B). We hypothesize that small molecules have broader inhibitory effects compared with shRNA knockdowns, targeting several kinases simultaneously. Indeed, dasatinib inhibits SRC kinases and LCK (39) and the SYK inhibitors R406/R778 have significant off-target effects including LCK (40, 41). Alternatively, shRNA knockdown vectors reduce the transcript levels of targeted genes without inhibiting the kinase activity directly and low kinase activity might be sufficient to sustain cell survival and proliferation. Hence, leukemia cells are more dependent on SYK than on LCK and ZAP70 as shown in vivo in mouse transplantation assays (Supplementary Fig. S3C) as well as in vitro in human cell lines (Supplementary Fig. S5B), suggesting different hierarchical dependencies among E2A-PBX1 target genes.
The cross-species comparative studied using genetic inactivation by shRNAs and pharmacologic inhibition by small molecules strongly suggests that ZAP70, SYK, LCK as well as PLCγ2 play a pathogenic role in E2A-PBX1 leukemogenesis. Our studies indicate that PLCγ2 is a key downstream protein, which mediates cell proliferation not only in E2A-PBX1+/preBCR+ but also in E2A-PBX1+/preBCR− and non-E2A-PBX1 ALLs. Although E2A-PBX1 leukemia cells have different levels of pPLCγ2, we did not correlate any characteristic with the activation of pPLCγ2. Particularly, we did not find any difference regarding PAX5 status, mouse Cre line employed, or preBCR expression. We hypothesize that preBCR− ALL might activate alternative pathways involved in proliferation and survival. Indeed, we described the acquisition of mutations leading to increased activation of the JAK/STAT and RAS/MAPK pathway in previous studies (5). Although ZAP70, LCK, and SYK are expressed in E2A-PBX1+/preBCR− leukemias, they maintain pPLCγ2 levels through alternative pathways that might result in resistance to the inhibitors as shown for human cell lines (Fig. 5A).
Our data suggest that the presence of the preBCR increases the dependence on the enzymatic activity of ZAP70, LCK, and SYK in leukemia cells without increasing substantially pPLCγ2 levels. We elucidated pathways upstream of PLCγ2 that are activated in E2A-PBX1+/preBCR+ leukemias and confer susceptibility to small-molecule inhibitors. Future studies are necessary to characterize activated pathways upstream of PLCγ2 in E2A-PBX1+/preBCR− as well as in B-ALLs driven by different oncogenes, which might reveal novel pathways for targeted therapies. Alternative pathways may be involved in PLCγ2 activation including receptor tyrosine kinases (42), Tec family kinases as BTK (43) and PI3 kinases (44).
In previous studies, PLCγ2 and ZAP70 were described as predictive markers for dasatinib response in CLL (45, 46) and PLCγ2 in diffuse large B-cell lymphoma (47).Our data suggest that PLCγ2 may be used as a predictive marker for dasatinib as well as SYK and LCK inhibitor responses in preBCR+ ALL, although prospective studies in primary ALLs are needed. ZAP70 shows promise as a possible therapeutic target in several hematologic malignancies, including CLL and preBCR+ ALL; however, ZAP70 inhibitors are currently not available. We suggest that the development of ZAP70 inhibitors will have an impact in preBCR+ ALL treatment, as single and also combination therapy.
In summary, signaling network remodeling by E2A-PBX1 induces aberrant phosphorylation of PLCγ2, consistent with activation of upstream signaling pathways. The E2A-PBX1 target genes ZAP70, SYK, and LCK encode kinases upstream of PLCγ2 and play an oncogenic role in pre-B-ALL. Small molecules including dasatinib, as well as SYK and LCK inhibitors, target signaling pathways upstream of PLCγ2 and show promising activity in vitro and in vivo for the treatment of preBCR+ ALLs.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: J. Duque-Afonso, M.L. Cleary
Development of methodology: J. Duque-Afonso, C.-H. Lin
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Duque-Afonso, K. Han, M.C. Wei, J.H. Kurzer, C. Schneidawind
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Duque-Afonso, C.-H. Lin, K. Han, M.C. Wei, C. Schneidawind, S.H.K. Wong, M.C. Bassik, M.L. Cleary
Writing, review, and/or revision of the manuscript: J. Duque-Afonso, M.C. Wei, C. Schneidawind, M.C. Bassik, M.L. Cleary
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.-H. Lin, J. Feng, C. Schneidawind, M.C. Bassik
Study supervision: J. Duque-Afonso, M.L. Cleary
We thank Maria Ambrus and Cita Nicolas for technical assistance, members of M.L. Cleary's laboratory for helpful discussions, and Carlos Duque-Afonso for graphic design.
This work was supported in part by grants from William Lawrence and Blanche Hughes Foundation (M.L. Cleary), the German Research Foundation (Deutsche Forschungsgemeinschaft, ref. DU 1287/2-1 to J. Duque-Afonso), the Lucile Packard Foundation for Children's Health, the Child Health Research Institute and the Stanford NIH-NCATS-CTSA grant #UL1 TR001085 (M.L. Cleary, J. Duque-Afonso, C.-H. Lin, J. Feng), Alex's Lemonade Stand Foundation for Childhood Cancer (S.H.K. Wong), and Dr. Mildred Scheel Fellowship of the German Cancer Aid (C. Schneidawind). K. Han was supported by The Walter V. and Idun Berry Postdoctoral Fellowship Program and M.C. Bassik was supported by a grant from Stanford ChEM-H and an NIH Directors New Innovator Award (1DP2HD08406901).
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