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. 2015 Sep;47(9):1020-1029.
doi: 10.1038/ng.3362. Epub 2015 Jul 27.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options

Ute Fischer #  1 , Michael Forster #  2 , Anna Rinaldi #  3 , Thomas Risch #  4 , Stéphanie Sungalee #  5 , Hans-Jörg Warnatz #  4 , Beat Bornhauser  3 , Michael Gombert  1 , Christina Kratsch  6 , Adrian M Stütz  5 , Marc Sultan  4 , Joelle Tchinda  3 , Catherine L Worth  4 , Vyacheslav Amstislavskiy  4 , Nandini Badarinarayan  2 , André Baruchel  7 , Thies Bartram  8 , Giuseppe Basso  9 , Cengiz Canpolat  10 , Gunnar Cario  8 , Hélène Cavé  11 , Dardane Dakaj  3 , Mauro Delorenzi  12   13 , Maria Pamela Dobay  13 , Cornelia Eckert  14 , Eva Ellinghaus  2 , Sabrina Eugster  3 , Viktoras Frismantas  3 , Sebastian Ginzel  1   15 , Oskar A Haas  16 , Olaf Heidenreich  17 , Georg Hemmrich-Stanisak  2 , Kebria Hezaveh  1 , Jessica I Höll  1 , Sabine Hornhardt  18 , Peter Husemann  1 , Priyadarshini Kachroo  2 , Christian P Kratz  19 , Geertruy Te Kronnie  9 , Blerim Marovca  3 , Felix Niggli  3 , Alice C McHardy  6 , Anthony V Moorman  17 , Renate Panzer-Grümayer  16 , Britt S Petersen  2 , Benjamin Raeder  5 , Meryem Ralser  4 , Philip Rosenstiel  2 , Daniel Schäfer  1 , Martin Schrappe  8 , Stefan Schreiber  2 , Moritz Schütte  20 , Björn Stade  2 , Ralf Thiele  15 , Nicolas von der Weid  21 , Ajay Vora  22 , Marketa Zaliova  19   23 , Langhui Zhang  1   24 , Thomas Zichner  5 , Martin Zimmermann  19 , Hans Lehrach  4   20   25 , Arndt Borkhardt  1 , Jean-Pierre Bourquin  3 , Andre Franke  2 , Jan O Korbel  5 , Martin Stanulla  19 , Marie-Laure Yaspo  4
Affiliations

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options

Ute Fischer et al. Nat Genet. 2015 Sep.

Abstract

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

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Figures

Figure 1
Figure 1. Genetic lesions identified in pediatric TCF3-HLF- and TCF3-PBX1-positive ALL
(a) Breakpoints in TCF3, PBX1 and HLF cluster in genomic hotspot regions. Boxes correspond to exonic regions; arcs represent fusions in patient samples. (b) TCF3 breakpoints cluster in two TCF3 intronic regions: between exons 16 and 17 (type 1) and between exons 15 and 16 (type 2). On the transcript level, type 1 translocations join TCF3 exon 16 to HLF exon 4, including inserted non-template and intronic sequences and new splice acceptor sites (patients 8 and 9). Type 2 translocations occur downstream of exon 15 and exclude TCF3 exon 16 from the fusion transcript (patients 6, 7 and 11). (c) Absence of overlapping somatic structural and nucleotide variations between the cohorts. TCF3-HLF-positive ALL is characterized by mutually exclusive PAX5, BTG1 and VPREB1 deletions and nonsynonymous nucleotide variations in TCF3 (p.Asp561Val, “D561V” in patient 8 and p.Ser467Gly in patient 13 of the validation cohort, the latter not included here). Subclonal RAS pathway mutations are exclusively detected in HLF-, but not in PBX1-fused cases. (d) Models of wild-type and mutant TCF3 based on the crystal structure of TCF3 in complex with the transcription factors SCL, LMO2 and LDB1 bound to DNA. Upon LMO2 binding, bonds are formed between TCF3 and SCL, including a hydrogen bond (dashed line) between D561 and R230 reducing the DNA binding capacity of the complex. Inset: D561V introduces a hydrophobic valine residue close to polar residues that may interfere with hydrogen bonding, thus altering the DNA binding properties of the complex. (For details see Source file for Figure 1.)
Figure 2
Figure 2. TCF3-HLF programs leukemia to a hybrid hematopoietic transcriptional state
(a) Heatmap of the 401 differentially expressed genes between the two ALL subtypes (edgeR, |log2(fold change)| ≥ 1, FDR ≤ 0.001). (b) Enriched hematopoietic stages in TCF3-HLF-positive (orange) and TCF3-PBX1-positive (green) ALL. Stages shown include hematopoietic stem cells (HSC), common myeloid progenitors (CMP), lymphoid-specified progenitors (GMP and MEP), neutrophils (NEUTRO), monocytes (MONO), multilymphoid progenitor (MLP), early T cell precursors (ETP), pro-B cells (PROB), T cells (TCELL), and B cells (BCELL). Gene set enrichment analysis was carried out using Genomatix genome analyzer and Gene Set Enrichment Analysis (GSEA) (GSEA: FDR ≤ 0.02; genomatix genome analyzer: adjusted p-value ≤ 0.02). The source of the significant enriched gene sets is noted by the superscript: 1) curated gene sets of hematopoietic precursors; 2) human immunologic gene signatures (MSigDB v4.0); 3) text mining-based tissue specific gene sets. (c) Enrichment plot for the HSC signature. (d) Components of the TCF3-HLF-positive ALL signature reveal functional annotation related to stem cells and their cellular location (Genomatix genome analyzer: p-value = 4.65 × 10−4, adjusted p-value < 0.001). (For details see Source file for Figure 2.)
Figure 3
Figure 3. The genomic landscape of TCF3-HLF- and TCF3-PBX1-positive ALL is preserved in patient-derived leukemia xenografts
(a) Xenografts were established from cryopreserved patient samples at diagnosis (samples “a”), at follow-up with minimal residual disease (MRD, <1 leukemic cell in 10,000 cells, samples “b”) or from disease progression (samples “c”) and subjected to whole exome and transcriptome sequencing as well as multiplex ligation-dependent probe amplification (MLPA). All available MRD samples from TCF3-HLF-positive cases were successfully engrafted. (b) Comparison of all transcriptionally expressed nucleotide variations and of selected recurrent deletions frequently found in pediatric ALL in corresponding patient (P) and xenograft (X) samples. Deletions and nucleotide variations are colored according to their frequency in the analyzed leukemic cell population. Deletion frequencies were calculated by integrating whole genome and whole exome sequencing data with MLPA data. Nucleotide variation frequencies were calculated by integrating whole genome, whole exome and transcriptome sequencing data. (For details see Source file for Figure 3.)
Figure 4
Figure 4. Major components of the gene expression signature of TCF3-HLF- and TCF3-PBX1-positive ALL are conserved in patient-derived xenografts
Hierarchical clustering of primary and patient derived xenograft (PDX) ALL samples based on the expression of the 401 genes of the signature defined with primary samples (Fig. 2) shows that xenografts clearly group with their corresponding primary samples. (For details see Source file for Figure 4.)
Figure 5
Figure 5. Drug activity profiling of TCF3-translocated leukemia reveals relevant differences in drug sensitivity
(a) Unsupervised clustering based on the drug activity profile of 98 compounds (logIC50) separates the two subtypes. Fitted values are provided in the supplementary section (absolute IC50). Numbers identify the compounds shown in (c). (b) Principal component analysis of the response variables IC50, EC50, EC90 and AUC (Supplementary Table 24) show TCF3-PBX1-positive and TCF3-HLF-positive ALL in two distinct clusters. The separation of TCF3-PBX1-positive and TCF3-HLF-positive ALL is determined by responses to topoisomerases, BCL2 inhibitors, glucocorticoids and antimitotic agents, which correlate with the first three principal components. (c) Selection of drugs based on differences in sensitivity or resistance in TCF3-PBX1-positives and TCF3-HLF-positives. For comparison, the corresponding drug activity is indicated for 25 additional ALL samples tested on the same platform, including standard risk (SR, n=5), medium risk (MR, n=4), and high risk (HR, n=16) cases (Supplementary Table 25). Boxplots extend from the first to the third quartiles (hinges) of the response range for each compound. Whiskers correspond to values from the hinge to the lowest or highest values within 1.5x of the distance between the first and third quartiles, respectively. Drugs with differential activity include docetaxel, paclitaxel, vincristine, AT9283, barasertib, BI2536, torin-1, dasatinib, lestaurtinib and XL228 (p≤0.05). Drugs which are active across the patients include doxorubicin, idarubicin, mitoxantrone, bortezomib, panobinostat, NVP-AUY922, ABT-199 (venetoclax) and navitoclax. (For details see Source file for Figure 5.)
Figure 6
Figure 6. The BCL2 antagonist ABT-199 (venetoclax) shows promising anti-leukemic activity in TCF3-HLF-positive xenografts
(a) In vitro dose response curves normalized against DMSO treated controls. (b) Merged absolute RPKM values of xenografts derived from the same primary leukemia sample (upper panel) and western blot for BCL2 (lower panel) in patient-derived xenografts as indicated. (c,d) In vivo response to ABT-199 on TCF3-HLF-positive xenografts. Treatment (grey bar) with 100 mg kg-1 qd ABT-199 (magenta) or with vehicle control (turquoise) were administered orally for 14 days (6-8 mice per treatment arm). Two treatment courses were administrated to xenograft 7a. For survival analysis an event was defined when at least 25% of leukemic cells were detected by FACS (mCD45−hCD19+hCD45+) in the peripheral blood. Differences in the survival of mice receiving ABT-199 or vehicle control were determined by the Mantel-Cox test and verified by the Gehan-Breslow-Wilcoxon test. (e) Mice from the control arm of (c,d) were treated with ABT-199 when more than 50% of ALL cells were detected in the blood. Mean and SD are shown (n=4). (For details see Source file for Figure 6.)
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