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

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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Figure 1: Genetic lesions identified in pediatric TCF3-HLF– and TCF3-PBX1–positive ALL.
Figure 2: TCF3-HLF programs leukemia to a hybrid hematopoietic transcriptional state.
Figure 3: The genomic landscape of TCF3-HLF– and TCF3-PBX1–positive ALL is preserved in patient-derived leukemia xenografts.
Figure 4: Major components of the gene expression signature of TCF3-HLF− and TCF3-PBX1–positive ALL are conserved in patient-derived xenografts.
Figure 5: Drug activity profiling of TCF3-translocated leukemia reveals relevant differences in drug sensitivity.
Figure 6: The BCL2 antagonist ABT-199 (venetoclax) shows promising anti-leukemic activity in TCF3-HLF–positive xenografts.

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Acknowledgements

We thank all participants and personnel involved in the clinical trials in Austria, France, Germany, United Kingdom and Switzerland. We thank T. Radimerski and Novartis for providing essential compounds. We thank the Leukaemia & Lymphoma Research (LLR) Childhood Leukaemia Cell Bank in the UK for providing primary patient samples. This work was supported by the German Federal Office for Radiation Protection (grant St.Sch. 3611S70014), by the Swiss National Research Foundation SNF 310030-133108, the foundation 'Kinderkrebsforschung Schweiz', the 'Krebsliga Zurich', the Sassella Foundation, the Fondation Panacée, the clinical research focus program 'Human Hemato-Lymphatic Diseases' of the University of Zurich, the Deutsche Forschungsgemeinschaft (DFG), Clusters of Excellence 'Inflammation at Interfaces', the EU Seventh Framework Program (FP7/2007-2013, grant 262055, ESGI; FP7-HEALTH-F2-2011 grant 261474, ENCCA; ERA-Net Transcan, Validation of biomarkers for personalized cancer medicine, TRANSCALL; Health-F2-2010 grant 260791, EUROCANPLATFORM), the 'Katharina Hardt Stiftung', the 'Deutsche José Carreras Leukämie-Stiftung', the 'Madeleine Schickedanz-Kinderkrebs-Stiftung', the 'Deutsche Krebshilfe – Dr. Mildred Scheel Stiftung' (grants 108613, 102588 and 108588), the Foundation of Experimental Biomedicine in Zurich, the Max Planck Society, and the 'Verein für krebskranke Kinder Hannover e.V.'. We thank A. Dehos, B. Grosche, T. Jung, W. Weiss and G. Ziegelberger, German Federal Office for Radiation Protection, as well as B. Heinzow, State Office for Social Services of Schleswig-Holstein, and A. Böttger, German Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety for their support and critical discussions. We are grateful for the excellent technical assistance offered by the sequencing team of the Department of Vertebrate Genomics of the Max Planck Institute for Molecular Genetics (Berlin) and by the team of the Genomics Core facility of the European Molecular Biology Laboratory. We thank K. Alemazkour for excellent technical assistance regarding whole exome sequencing at the Department of Pediatric Oncology, Hematology and Clinical Immunology (Düsseldorf, Germany). We thank N. Forgo, Institute for Legal Informatics, Leibniz University Hannover, and H.-D. Tröger, Hannover Medical School, for legal and ethical counselling.

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Authors and Affiliations

Authors

Contributions

A. Borkhardt, A.F., J.O.K., J.-P.B., M.-L.Y. and M. Stanulla jointly designed the project. A. Baruchel, A.V., A.V.M., C.C., C.P.K., F.N., G.B., G.C., G.t.K., H.C., M. Schrappe, M. Stanulla, N.v.d.W., O.A.H., C.E. and R.P.-G. provided samples or clinical data. A. Borkhardt, A.C.M., A.F., A.V., C.E., G.H.-S., H.L., J.-P.B., J.O.K., J.T., M.D., M.F., M.-L.Y., M.P.D., M. Schrappe, M. Stanulla, M. Zimmermann, O.A.H., P.R. and T.Z. contributed reagents, materials or analysis tools. A.M.S., B.B., B.M., C.E., C.L.W., H.-J.W., J.I.H., J.-P.B., M.F., M.G., S. Sungalee and U.F. designed experiments. A.M.S., A.R., B.B., B.R., B.S., B.S.P., C.E., C.K., C.L.W., D.D., D.S., H.-J.W., J.T., K.H., L.Z., M.G., M.R., M. Zaliova, M. Sultan, P.K., S.E., S. Sungalee, T.B., U.F. and V.F. performed experiments. A.R., B.S., B.S.P., C.E., C.K., C.L.W., D.S., E.E., J.I.H., H.-J.W., M.G., M.P.D., M.F., N.B., S.G., G.H.-S., P.H., P.K., M.-L.Y., M.R., M. Stanulla, M. Schütte, M. Zaliova, S. Sungalee, T.R., U.F. and V.A. analyzed data. A. Borkhardt, A.C.M., A.F., B.B., H.L., J.-P.B., J.O.K., M.D., M.F., M.-L.Y., M. Stanulla, O.H., R.T. and S. Schreiber, U.F. supervised research. A.R., C.L.W., D.S., H.-J.W., M.F., M.P.D., M. Stanulla, P.K., S. Sungalee, T.R. and U.F. prepared tables and figures. J.-P.B., M. Stanulla and M.-L.Y. wrote the manuscript. A. Borkhardt, A.F., A.R., A.V.M., H.-J.W., J.O.K., M.F., M.P.D., O.H., S.H., S. Sungalee, T.R. and U.F. contributed to the writing of the manuscript. All authors critically reviewed the manuscript for its content.

Corresponding authors

Correspondence to Jean-Pierre Bourquin or Martin Stanulla.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 TCF3 breakpoints of TCF3-PBX1 (patients 1a−5a) and TCF3-HLF (patients 6a−9a and11a) translocations.

The CpG motifs closest to the breakpoints are highlighted in red boxes and the closest cac motifs in blue.

Supplementary Figure 2 The TCF3-HLF translocation is only detected in the lymphoid progenitor gate.

Experiments, combining sorting of specific cell populations using flow cytometry (a) and subsequent detection of the fusion gene in sorted cells employing FISH probes or real-time PCR (b), were performed using bone marrow from the TCF3-HLF-positive patients 15, 16, and 17 of the validation cohort. Bone marrow cells were sorted into HSC (CD34+CD38-CD90+CD45RA-) and MLP (CD34+CD38-CD90neg-lowCD45RA+) populations as previously described (Doulatov et al. Nature Immunology 11, 585-593 (2010)). Leukemic cells (CD34-CD38+CD19+CD10+) were sorted according to standard diagnostic markers. Sorted cell populations were analyzed by FISH and real-time PCR as described in the Online Methods section. All sorted leukemia cells carried the translocation (FISH signals 2R, 1G, 1B, 2B/G fusion) while none of the scored HSC or MLP (2R, 2G, 2B) populations were positive. The TCF3-HLF fusion was detected only in blast populations and not in myeloid cells indicating that the TCF3-HLF-positive leukemia originated in committed lymphoid cells. For patient 15, the results were confirmed using a TCF3-HLF-fusion-specific quantitative real-time PCR on a fraction of sorted cells as described in the supplementary online methods section. Leukemic cells were 100% positive for the TCF3-HLF-fusion, whereas the MLP and the HSC fractions were negative. As a positive control the reference gene β-globin was employed. The TCF3-HLF fusion was detected by real-time PCR only in the blast population, but not in myeloid cells, indicating that the TCF3-HLF-positive leukemia may have originated from committed lymphoid cells.

Supplementary Figure 3 BTG1 deletions.

(a) Whole genome sequencing (WGS) read ratio plot showing a BTG1 deletion identified in the diagnostic sample of patient 8. This BTG1 deletion was present in a subclonal population of ~14% of leukemia cells. (b) WGS read ratio plot showing a BTG1 deletion identified in the diagnostic sample of patient 9. (c) Exome sequencing read ratio plot for a xenograft (7b_X) derived from the remission sample of patient 7 showing a BTG1 deletion. (d) The BTG1 deletion in xenograft 7b_X ia also visible in the SNP allele frequency data from exome sequencing, inspecting the SNP rs709222 in BTG1 exon 2.

Supplementary Figure 4 KHDRBS1-LCK fusion gene in patient 6a.

(a) A heterozygous deletion encompassing 223 kb of genomic DNA on chromosome 1 fused intron 1 of KHDRBS1 to the promoter region of the LCK proto-oncogene. (b) Transcriptome sequencing showed a decrease of KHDRBS1 expression after exon 1. (c) LCK expression was induced starting from exon 2. (d) Analysis of chimeric fusion reads showed the fusion of KHDRBS1 exon 1 with LCK exon 2, confirmed by 14 chimeric reads spanning the fusion border. (e) Amino acid sequence of the in-frame fusion protein encoded by the KHDRBS1-LCK fusion transcript.

Supplementary Figure 5 Recurrent fusion of the genes KHDRBS1 and LCK in ALL.

RNA was isolated from samples of a cohort of 74 unselected pediatric ALL and transcribed into cDNA. Nested PCR was performed using specific primers for the KHDRBS1-LCK fusion transcript detected in patient 6a. (a) The gel electrophoresis presents the expected specific PCR product of 226 bp in the sample 6a and three patient samples of the validation cohort as well as controls. The negative control is representative of the 71 patient samples of this validation cohort tested negative. (b) The chromatograms display the sequencing result for patient 6a (upper panel) and a patient of the validation cohort (patient D3, lower panel) as an example. The fusion of KHDRBS1 exon 1 to LCK exon 2 was confirmed by Sanger sequencing in all positive cases (3/74).

Supplementary Figure 6 Pattern of recurrent genomic lesions and model of leukemogenesis in TCF3-HLF−positive leukemia.

Left panel: Recurrent genetic lesions identified in TCF3-HLF-positive ALL affect transcription factors regulating lymphoid differentiation, the Ras pathway and cell cycle regulation. Besides the 12 cases analyzed in the present study, a single case reported in a recent study is included (Ma et al. Nature Communications 6, 6604 (2015), DOI: 10.1038/ncomms7604) (x indicates a KRAS mutation that was detected only in a patient derived xenograft sample, not in the primary tissue. Note that the cases 16 and 17 have not been analyzed by next generation sequencing and besides PAX5 deletion, NRAS and KRAS hotspot mutations other genetic alterations possibly present are not yet analyzed). Right panel: The TCF3-HLF gene fusion probably occurs in an early B cell progenitor. B cell differentiation is blocked by mutations in transcription factors playing key roles in lymphoid development. Secondary mutations involving the Ras pathway and cell cycle regulators lead to leukemia development.

Supplementary Figure 7 Engraftment of TCF3-PBX1− and TCF3-HLF−positive ALL samples.

ALL cells were recovered from cryopreserved samples and transplanted into NSG mice. The percentage of mCD45-hCD19+hCD45+ leukemic cells was monitored by flow cytometry every 4 weeks. Upper panels: Primary and secondary transplantation of diagnostic TCF3-PBX1-positive samples. Middle panels: Primary and secondary transplantation of diagnostic TCF3-HLF-positive samples. Lower panels: Primary and secondary transplantation of MRD and relapse TCF3-HLF-positive samples. MRD samples were successfully amplified only for the TCF3-HLF-positive subtype. Patient samples were transplanted intrafemorally (primary transplantation). Xenograft-samples were transplanted intravenously (secondary transplantation). Mean and SD of 2-3 mice is shown when available. The number of living cells transplanted is indicated in the legend.

Supplementary Figure 8 Expression of PAX5 in primary and patient derived xenografts (PDX) of TCF3-PBX1− and TCF3-HLF−positive ALL samples.

The significant differential expression of PAX5 between TCF3-PBX1-positive and TCF3-HLF-positive primary ALL is preserved in patient derived xenograft samples.

Supplementary Figure 9 Cell survival and cell cycle analysis of TCF3-PBX1− and TCF3-HLF−positive ALL cells in coculture on hMSCs.

Upper panel: ALL cell viability comparing co-cultures to ALL cell suspension cultures after 72 h by flow cytometry using 7-AAD staining. The percentage of living cells normalized to the input cells seeded on day 0 is shown (mean with SD of duplicates). Lower panel: Percentage of cells of the population in the G0–G1, S and G2–M phases of the cell cycle. Flow cytometric analysis was performed at day 1 of co-culture with hMSC after 20 h of EdU incubation. Mean and SD of at least 2 independent experiments performed in triplicates are shown.

Supplementary Figure 10 Drug profiling of TCF3-PBX1 and TCF3-HLF ALL xenografts derived from diagnostic, MRD and relapse samples.

Unsupervised clustering of xenograft samples according to their response (log IC50) to 98 compounds in co-culture on hMSC after 72 h. Xenografts derived from diagnostic „a“, MRD „b“ and relapse „c“ samples are included. Fitted values are provided in the supplementary section (absolute IC50). Compounds are arranged based on the family of the targets. Numbers identify the compounds shown in Figure 5c.

Supplementary Figure 11 In vitro dose response curves to ABT-199 (venetoclax) comparing xenografts derived from TCF3-HLF-positive ALL patients at the time point of diagnosis 'a', MRD 'b' and relapse 'c'.

ALL co-cultures were treated with ABT-199 for 72 hours, the responses were normalized against DMSO-treated controls.

Supplementary Figure 12 Drug activity profiling of two additional TCF3-HLF−translocated leukemia cases of the validation cohort (15, 17) reveals similar patterns of drug sensitivities and high responsiveness to ABT-199 (venetoclax) compared to the TCF3-HLF−positive patients of the discovery cohort.

Selection of drugs based on differences in sensitivity or resistance in TCF3-PBX1-positive compared to TCF3-HLF-positive patients (Figure 5c). Similar patterns of drug sensitivity were observed testing patient derived primary (patient 15, 17) and xenograft cells (other TCF3-PBX1- and TCF3-HLF-positive cases shown). Drugs active across both patient cohorts include doxorubicin, idarubicin, mitoxantrone, bortezomib, and ABT-199 (venetoclax).

Supplementary Figure 13 In vitro dose response co-titration curves to ABT-199/dexamethasone (top) and ABT-199/vincristine (bottom) for patient derived xenografts from TCF3-HLF−positive (6a−11a) ALL patients.

Xenograft cells were seeded on a layer of stroma cell and treated with the indicated combinations and concentrations of ABT-199, vincristine and dexamethasone, respectively, for 72 h. Drug concentrations used in these assays are provided in Supplementary Table 26. The response was normalized against DMSO-treated controls. The co-titration curves indicate that ABT-199 combined with either vincristine or dexamethasone likely exhibits synergistic activity in the patients marked with (*); combination indices (see Table) were calculated based on the Chou-Talaly method (Chou and Talaly JBC 252: 6438-6442, (1977)).

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Fischer, U., Forster, M., Rinaldi, A. et al. Genomics and drug profiling of fatal TCF3-HLF−positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options. Nat Genet 47, 1020–1029 (2015). https://doi.org/10.1038/ng.3362

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