Complete hematologic response of early T-cell progenitor acute lymphoblastic leukemia to the γ-secretase inhibitor BMS-906024: genetic and epigenetic findings in an outlier case

Abstract

Notch pathway antagonists such as γ-secretase inhibitors (GSIs) are being tested in diverse cancers, but exceptional responses have yet to be reported. We describe the case of a patient with relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) who achieved a complete hematologic response following treatment with the GSI BMS-906024. Whole-exome sequencing of leukemic blasts revealed heterozygous gain-of-function driver mutations in NOTCH1, CSF3R, and PTPN11, and a homozygous/hemizygous loss-of-function mutation in DNMT3A. The three gain-of-function mutations were absent from remission marrow cells, but the DNMT3A mutation persisted in heterozygous form in remission marrow, consistent with an origin for the patient's ETP-ALL from clonal hematopoiesis. Ex vivo culture of ETP-ALL blasts confirmed high levels of activated NOTCH1 that were repressed by GSI treatment, and RNA-seq documented that GSIs downregulated multiple known Notch target genes. Surprisingly, one potential target gene that was unaffected by GSIs was MYC, a key Notch target in GSI-sensitive T-ALL of cortical T-cell type. H3K27ac super-enhancer landscapes near MYC showed a pattern previously reported in acute myeloid leukemia (AML) that is sensitive to BRD4 inhibitors, and in line with this ETP-ALL blasts downregulated MYC in response to the BRD4 inhibitor JQ1. To our knowledge, this is the first example of complete response of a Notch-mutated ETP-ALL to a Notch antagonist and is also the first description of chromatin landscapes associated with ETP-ALL. Our experience suggests that additional attempts to target Notch in Notch-mutated ETP-ALL are merited.

Keywords: leukemia.

Figure 1.

Bone marrow biopsy histology prior to and 4 and 7 wk after initiation of treatment with BMS-906024 (hematoxylin and eosin staining, 400× magnification). The marrow contains mainly blasts prior to treatment that are cleared following treatment.

Figure 2.

Characterization of the NOTCH1 gain-of-function mutation F1592C. (A) Position of F1592C in the NOTCH1 negative regulatory region (NRR). Mapping is based on the crystallographic structure of Gordon et al. (2009b). Residues previously reported be mutated in T-ALL are shown in green (core residues), orange (interdomain interface residues), or purple (polar residues). The three LNR repeats are colored various shades of pink with a translucent surface, and the core “heterodimerization domain” is colored blue prior to the site of furin cleavage (HD-N) and cyan thereafter (HD-C). F1592 sits in the center of the hydrophobic core of the heterodimerization domain. The image is rendered with the program Pymol and is adapted from Gordon et al. (2009a). (B) F1592C leads to ligand-independent activation of NOTCH1. The ability of the F1592C variant to activate a Notch firefly luciferase reporter gene was compared with wild-type NOTCH1 and two previously characterized gain-of-function mutants, F1592S and L1593P. Firefly luciferase activity was normalized to an internal Renilla luciferase control reporter gene. Normalized firefly luciferase activity is expressed related to the activity of wild-type NOTCH1, which is arbitrarily set to a value of 1. Each expression plasmid was tested in three independent experiments; error bars represent the standard deviations. (C) Copy-number gains and losses. The copy number of chromosomal regions was determined from sequencing data as described (Wagle et al. 2012). (D) Effect of the GSI diaminobenzidine (DBZ) on activated NOTCH1 (ICN1) levels in leukemic blasts. Blasts were treated with vehicle (DMSO [dimethyl sulfoxide]) or DBZ for 3 d prior to harvest and preparation of whole-cell lysates. A representative Western blot stained for activated NOTCH1 (ICN1) and β-actin is shown.

Figure 3.

Evidence of ETP-ALL origin from clonal hematopoiesis. (A) Sanger sequencing traces of DNMT3A. Traces were obtained from DNA obtained from blasts prior to treatment, remission marrow, and saliva following hematopoietic stem cell transplantation. (B) Reconstructed clonal evolution of ETP-ALL, based on sequencing of DNMT3A as shown in A.

Figure 4.

Characterization of likely Notch target genes and MYC in ETP-ALL. (A) Genes downregulated by the GSI DBZ (1 μm) in ETP-ALL blasts. Cells were treated with DBZ or control (DMSO) for 3 d followed by harvest and analysis by RNA sequencing. FPKM represents the log₂ value of the FPKM (fragments per transcript kilobase per million fragments mapped). (B) Effect of the GSI DBZ on MYC levels in leukemic blasts. Blasts were treated with vehicle (DMSO) or DBZ for 3 d prior to harvest and preparation of whole-cell lysates. A representative Western blot stained for MYC and β-actin is shown. (C) H3K27ac ChIP-seq landscapes near MYC. Aligned reads are shown for leukemic blasts (Pt) and three NOTCH1-mutated T-ALLs, DND41, KOPT-K1, and CUTLL1, that downregulate MYC in response to GSI treatment. (NDME) Notch-dependent MYC enhancer; (BDME) BRD4-dependent MYC enhancer. (D) Effect of the bromodomain inhibitor JQ1 on MYC levels in leukemic blasts. Blasts were treated with vehicle (DMSO) or JQ1 for 3 d prior to harvest and preparation of whole-cell lysates. A representative Western blot stained for MYC and β-actin is shown.

Figure 5.

Characterization of ETP-ALL grafts in NSG-S mice (NSG mice expressing IL-3, GM-CSF, and KIT ligand). (A) Bone marrow histology (hematoxylin and eosin staining, 400× magnification). (B) Immunohistochemical staining for activated NOTCH1 (ICN1). Brown nuclear color denotes specific staining; hematoxylin was used as a counterstain.