doi: 10.3324/haematol.2023.283928.
Proteogenomic profiling uncovers differential therapeutic vulnerabilities between TCF3::PBX1 and TCF3::HLF translocated B-cell acute lymphoblastic leukemia
Affiliations
- PMID: 38426288
- PMCID: PMC11215373
- DOI: 10.3324/haematol.2023.283928
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Proteogenomic profiling uncovers differential therapeutic vulnerabilities between TCF3::PBX1 and TCF3::HLF translocated B-cell acute lymphoblastic leukemia
Haematologica.
.
No abstract available
Figures
Proteomic profiling distinguishes TCF3::HLF+
and TCF3::PBX1+ leukemia and uncovers therapeutic vulnerabilities for both subtypes. Unsupervised hierarchical clustering (A), principal component analysis (PCA) (B) and gene set enrichment analysis (GSEA) (C) was performed on the proteomic data of 6 TCF3::HLF+
(in red) and 5 TCF3::PBX1+ (in orange) B-cell acute lymphoblastic leukemia (B-ALL) patient-derived xenograft samples. For unsupervised hierarchical clustering, the 10% most variable proteins were used based on the standard deviation. PCA was performed on all proteins. Both subtypes clearly segregate into distinct clusters suggesting highly distinct proteomic landscapes. (C) GSEA is based on all proteins and identifies several gene sets enriched in either of the two subgroups. (D) Proteomic data shows enrichment of MYC protein expression in TCF3::HLF+ versus TCF3::PBX1+ leukemic samples in this study. In order to determine differential expression, non-parametric Mann-Whitney t test (two-tailed) was used. (E) Gene set enrichment plot of MYC targets showing a positive correlation with TCF3::HLF+
leukemia.
High-throughput drug screening and functional analysis confirm therapeutic vulnerabilities for TCF3::HLF+ and TCF3::PBX1+
leukemic cells. (A) HAL-01 (TCF3::HLF+), 697 and RCH-ACV (both TCF3::PBX1+) were exposed to a drug library, consisting of over 600 compounds. The supervised heatmap is based on the area under the curve (AUC, minimal fold change of <0.8 or >1.2 as a cutoff) as response parameter. Presented are groups of drugs with a differential response between TCF3::PBX1+ and TCF3::HLF+ leukemic cells. High sensitivity is shown in blue and low sensitivity is shown in white. (B-E) Presented are the dose-response curves for ixabepilone (B), SNS-032 (C), bleomycin sulfate (D) and idasanutlin (E), showing a differential response of TCF3::HLF+ versus TCF3::PBX1+ leukemic cells. Human peripheral blood cells from 3 healthy donors (in black) served as control cells (B-E). (F-I) RCH-ACV and HAL-01 were treated with ixabepilone (10 nM, 20 nM, 40 nM), SNS-032 (133 nM, 266 nM, 532 nM), idasanutlin (131 nM, 262 nM, 524 nM) or bleomycin sulfate (235 nM, 470 nM, 940 nM) dissolved in dimethyl sulfoxide (DMSO) or DMSO as negative control. (F, G) Bar graphs representing the fold change in caspase 3/7 activity 24 hours after treatment. Caspase 3/7 activity was determined using the Caspase-Glo 3/7 Assay (Promega). (H, I) Bar graphs represent the proportion of apoptotic cells (subG1) 48 hours after treatment. Cell cycle profiles were generated by flow cytometric measurement of propidium iodide intercalation into DNA after partial cell lysis in hypotonic buffer (0.1% sodium citrate, 0.1% Triton X-100, 0.5 mg/mL RNase A containing 40 µg/mL propidium iodide). Values shown in (F-I) represent mean ± standard error of the mean of 3 biologically independent replicates. NS: not significant; *P<0.05; **P<0.01; ***P<0.001 (t test).
Proteogenomic profiling detects BLK as a marker of TCF3::PBX1+ B-cell acute lymphoblastic leukemia targetable by the selective BLK inhibitor BLK-IN-2. (A) The volcano plot presents significantly (red) or non-significantly (grey) dysregulated proteins in TCF3::PBX1+ (left) or TCF3::HLF+ (right) leukemias. BLK is the most significantly dysregulated protein for the TCF3::PBX1+ subtype (minimal log2 fold change of ±1 and significance level of P<0.05 as cutoffs). (B) Gene expression data of human patient samples is derived from data sets available at the R2: genomics analysis and visualization platform (http://r2.amc.nl ). Dot plot presents PBX1 and BLK expression in healthy controls (black), TCF3::PBX1+ (orange) and other unstratified leukemic samples (grey). Data of the Mixed Leukemia - MILE - 2004 - MAS5.0 - u133p2 study is shown. Three further studies are presented in the Online Supplementary Figure S1D-F. (C,D) BLK RNA expression in TCF3::HLF+ (N=5) and TCF3::PBX1+ (N=5) B-ALL at the time of diagnosis (C) and after transplantation into NSG mice (TCF3::HLF+: N=22; TCF3::PBX1+: N=8). (D). Data is derived from our previous study. In order to determine differential expression, non-parametric Mann-Whitney t test (two-tailed) was used. (E) Dose-response curves for BLK-IN-2 show a differential response of TCF3::HLF+ (red) versus TCF3::PBX1+ (orange) leukemic cells. (F-H) Synergy drug screening of BLK-IN-2 and Ixabepilone reveals a strong synergistic effect in TCF3::PBX1+ cell lines, while no such effect is detected in the TCF3::HLF+ cell line HAL-01. Representative synergy plots of 3 independent experiments are shown. Drug concentration ranges were chosen according to the predetermined half-maximal inhibitory concentration (IC50) values of each cell line (RCH-ACV. and 697: 5-500 nM BLK-IN-2, 0.25-25 nM ixabepilone; HAL-01: 0.25-25 μM BLK-IN-2, 5-500 nM ixabepilone). Dimethyl sulfoxide (DMSO) was used as a negative control. ZIP synergy score analysis was conducted using the synergyfinder package version 3.0.14 with additional baseline correction.
References
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- Huang Y, Mouttet B, Warnatz HJ, et al. . The Leukemogenic TCF3-HLF complex rewires enhancers driving cellular identity and self-renewal conferring EP300 vulnerability. Cancer Cell. 2019;36(6):630-644. - PubMed
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