BCL6 enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-ABL1 kinase inhibition

Abstract

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.

Figure 1. Regulation of BCL6 expression in BCR-ABL1 ALL cells

a, Ph⁺ ALL cells were treated with and without Imatinib (10 μmol/l) for 24 hours. Upregulation of BCL6 was compared to expression levels in DLBCL with BCL6-IGH translocation by Western blot. b, BCR-ABL1-transformed mouse ALL cells were transduced with a constitutively active Stat5 mutant (STAT5-CA) or a control vector (GFP) and treated either with or without Imatinib. BCL6 Western blot was performed using β-actin as loading control. c, BCL6 expression upon Imatinib-treatment was studied by Western blot in the presence or absence of Cre-mediated deletion of Stat5 in BCR-ABL1-transformed Stat5^fl/fl mouse ALL. d, Mouse BCR-ABL1 ALL cells were transduced with FoxO4-Puromycin or a Puromycin control vector and subjected to antibiotic selection. Cells were harvested and BCL6 mRNA levels were measured by qRT-PCR relative to Hprt. e, Imatinib-induced BCL6 expression was studied by Western blot in the presence or absence of Cre-mediated deletion of Pten in BCR-ABL1-transformed Pten^fl/fl mouse ALL cells.

Figure 2. BCL6 is required for transcriptional inactivation of the Arf/p53 pathway in BCR-ABL1 ALL

a, Western blot analysis of CDKN2A (Arf) and p53 expression in BCL6^−/− and BCL6^+/+ BCR-ABL1 ALL cells. b, Human Ph⁺ ALL cells (Tom1) were treated with and without Imatinib (10 μmol/l) for 24 hours and were subjected to ChIP-on-chip analysis using a BCL6-specific antibody. The y-axis indicates enrichment versus input and the x-axis the location of probes within the respective loci relative to the transcriptional start site. The dark and light green (Control) or red (Imatinib) tracings depict two replicates. Recruitment to CDKN1A, CDKN1B, TP53 and HPRT (negative control) is shown in Ph⁺ ALL cells and one DLBCL cell line (OCI-Ly7). c, Cell cycle analysis (BrdU/7-AAD staining). d, Staining for senescence-associated β-galactosidase (SA-β-gal). ALL cells were treated with or without 0.05 μg/ml adriamycin for 48 hours to induce a low level of DNA damage. Percentages of SA-β-gal⁺ cells are indicated (means ± SD; n=3).

Figure 3. BCL6 is required for leukemia-initiation in BCR-ABL1 ALL

a, 10,000 BCL6^−/− or BCL6^+/+ BCR-ABL1 ALL cells were plated in semisolid agar, and colonies were counted after ten days (numbers denote means ± SD, n=3). b, Overall survival of mice injected with 100,000 BCL6^−/− and BCL6^+/+ BCR-ABL1 ALL cells was compared by Kaplan-Meier analysis. c, For a SCID leukemia-initiating cell (SL-IC) experiment, BCL6^−/− and BCL6^+/+ BCR-ABL1 ALL cells were labeled with firefly luciferase and were intravenously injected into sublethally irradiated NOD/SCID mice. Mice that developed CD45.1⁺ endogenous leukemia instead of leukemia from injected CD45.2⁺ cells are indicated by asterisks (see Supplementary Fig. 13). d, The SL-IC assay was repeated as a limiting dilution experiment (10³, 10⁴, 10⁵, 5 million cells) and leukemia cells were directly injected into the femoral bone marrow to circumvent potential engraftment defects.

Figure 4. BCL6 promotes survival of TKI-treated BCR-ABL1 ALL cells

a, Imatinib-sensitivity of BCL6^−/−, BCL6^+/− and BCL6^+/+ ALL cells was measured in a Resazurin viability assay. b, BCL6^−/− ALL cells were transduced with BCL6-ER^T2 or ER^T2 vectors (tagged with GFP). ALL cells were treated with or without 1 μmol/l Imatinib, and BCL6-ER^T2 or ER^T2 were induced by 4-OHT. Relative changes of GFP⁺ cells after induction are indicated. c, Patient-derived Ph⁺ ALL cells (ICN1) were transduced with inducible dominant-negative BCL6 (DN-BCL6-ER^T2) or ER^T2 control vectors. ALL cells were treated with or without 10 μmol/l Imatinib and DN-BCL6-ER^T2 or ER^T2 were induced by 4-OHT. Relative changes of GFP⁺ cells after induction are indicated. d, Patient-derived Ph⁺ ALL cells (TXL2) were labeled with luciferase and 100,000 cells were injected. Mice were treated 7 times with either vehicle (gray), Nilotinib (25 mg/kg; green) or a combination of Nilotinib and RI-BPI (25 mg/kg; red). Treated mice are shown in e, a Kaplan-Meier survival analysis. Treatment days are indicated by arrow heads.