Transcriptomic profiling reveals a correlation of cladribine responses with the expression of its target-gene RRM2. (A-D) The 20 primary T-PLL samples, which were subjected to the ex vivo single-agent cell-viability testing, were additionally characterized by polyA messenger RNA (mRNA) sequencing (mRNA-seq). (A-C) As the responses toward cladribine were the most heterogenous in the single-compound screening, we defined cladribine responders (DSS ≥10; n = 12 patients with T-PLL) and cladribine nonresponders (DSS <10; n = 8 patients with T-PLL). (A) Volcano plot showing fold-changes and adjusted P values of significantly deregulated genes when comparing cladribine responders and nonresponders. Upregulated genes in cladribine responders are shown in red and downregulated genes in blue (adjusted P < .05). (B) GSEA was performed for all differentially expressed genes between cladribine responders and nonresponders, applying HALLMARK gene sets. Significantly enriched pathways are displayed in the bar chart (adjusted P < .05; Kolmogorov-Smirnov test). (C) Heat map (left) presenting the patient-specific cladribine sDSS and RRM2 mRNA expression across the 20 T-PLL cases (R² = 0.56; P = .01, Pearson correlation). Bar chart (right) comparing the RRM2 mRNA expression between cladribine responders and nonresponders (mean with SEM; Student t test, ∗P < .05). Expression of the cladribine target-gene RRM2⁴⁰ correlated with the patient-specific sDSSs. (D) Case-specific color code (left) summarizing genomic alterations in ATM and JAK/STAT pathway genes, based on mRNA-seq results. Unsupervised hierarchical clustering is based on the ex vivo single-agent drug screening (see Figure 1B). Bar chart (right) showing the proportion of cases with a mutation and/or deletion in the respective gene. (dark blue, missense mutations; light-blue, nonsense mutations; blue, deletion; dark gray, multihit mutations; light gray, unmutated). (E) Baseline BH3 profiling in 10 of the 20 patient samples, that have been used in the initial ex vivo drug screen. Heat map (left) showing the relative cytochrome-c loss at baseline, as measured by flow cytometry (red, cytochrome-c release >0.5; blue, <0.5). Each row refers to a BH3 family peptide (incubation 1 hour), investigating either the overall priming (BIM and p53 upregulated modulator of apoptosis [PUMA]) or specific dependencies (Dep., BAD, HRK, MS1, and FS1). Correlation (right) between cytochrome-c release (x-axis) induced by BAD minus HRK (BAD – HRK = BCL2 dependence) and reduction of cell viability (y-axis) measured by CTG assay, after 72 hours exposure to 0.1 μM venetoclax (n = 10 cases). BCL2 dependence correlated with venetoclax-mediated reduction of cell viability (R² = 0.65; P = .005). See supplemental Figure 2 for patient-specific mRNA expression of T-PLL’s hallmark genes and drug target pathway genes. BIM, Bcl-2 interacting mediator of cell death; Dep., dependency; GSEA, gene set enrichment analysis; HRK, Harakiri, BCL2 interacting protein; MS1, non-natural MCL1-specific peptide 1; FS1, non-natural BFL1-specific peptide 1.