An immediate transcriptional signature associated with response to the histone deacetylase inhibitor Givinostat in T acute lymphoblastic leukemia xenografts

Abstract

Despite some success with certain hematological malignancies and in contrast with the strong pro-apoptotic effects measured in vitro, the overall response rate of acute lymphoblastic leukemia (ALL) to histone deacetylase inhibitors (HDACis) is low. With the aim to improve the understanding of how HDACis work in vivo, we investigated the therapeutic efficacy of the clinically approved HDACi Givinostat in a collection of nine pediatric human T-ALL engrafted systemically in NOD/SCID mice. We observed highly heterogeneous antileukemia responses to Givinostat, associated with reduction of the percentage of infiltrating blasts in target organs, induction of apoptosis and differentiation. These effects were not associated with the T-ALL cytogenetic subgroup. Transcriptome analysis disclosed an immediate transcriptional signature enriched in genes involved in cell-cycle regulation and DNA repair, which was validated by quantitative RT-PCR and was associated with in vivo response to this HDACi. Increased phospho-H2AX levels, a marker of DNA damage, were measured in T-ALL cells from Givinostat responders. These results indicate that the induction of the DNA damage response could be an early biomarker of the therapeutic effects of Givinostat in T-ALL models. This information should be considered in the design of future clinical trials with HDACis in acute leukemia.

Figure 1

Therapeutic effects of Givinostat in patient-derived T-ALL xenografts. (a) Outline of treatment with Givinostat (ITF2357) or vehicle (PEG400/H₂O). NOD/SCID mice (n=5/6 mice/group) were intraperitoneally treated with Givinostat (25 mg/kg) or vehicle 2 days after i.v. injection of T-ALL cells (5 × 10⁶cells/mouse). Givinostat was subsequently administered 5 days a week. Flow cytometric analysis of blood samples was used to track leukemia engraftment and progression. (b) Measurement of circulating blasts by flow cytometry after the last blood drawing (left panel, top) and quantification of infiltrating cells in the spleen (middle panel, top) and in the BM (right panel, top) at killing. Quantification of apoptotic leukemia cells in the spleen (left panel, bottom) and BM (middle panel, bottom). The spleen weight at killing was also reported (right panel, bottom). Results were expressed as mean value±S.D. Statistically significant differences are indicated (*P<0.05; **P<0.01; ***P<0.001). (c) Levels of acetylated α-tubulin were measured by western blotting analysis in PD-TALL8 (good responder), PD-TALL12 (partial responder) and PD-TALL9 (poor responder) cells obtained from the spleen of mice. A representative blot is shown. (d) Kaplan–Meier survival curves of mice engrafted with PD-TALL8 and PD-TALL16 after treatment with Givinostat or Vehicle (n=6 mice/group) (PD-TALL8: Log Rank P=0.0008; PD-TALL16: Log Rank P=0.0011)

Figure 2

Expression levels of TAL1 and TLX target genes. (a) Outline of treatment. Leukemic NOD/SCID (n=5/6 mice/group) were intraperitoneally treated once with Givinostat (25 mg/kg) or vehicle. Mice were killed 6 h after treatment. (b) T-ALL cells were recovered from the mice spleen and mRNA expression of several target genes were assessed by qRT-PCR. Results were expressed as mean value±S.D. Data were analyzed with Mann–Whitney test with Bonferroni correction (*P<0.05). (c) Leukemic cells were recovered from the spleen of PD-TALL12 mice and TLX1, TLX3 and TAL1 protein levels were analyzed by western blotting. Numbers below the bands indicate densitometric analysis of TLX1, TLX3 and TAL1 normalized to ACTIN. (d) Columns report the mean values±S.D. of TLX1, TLX3 and TAL1 ratios in control and treated mice (*P<0.05)

Figure 3

Givinostat induced differentiation of a TLX1-driven xenograft. (a) Flow cytometry analysis of CD1a (top) and CD4/CD8 (bottom) expression in human CD45-positive spleen cells isolated from PD-TALL8 leukemia recipient mice treated with Givinostat or vehicle (n=8/9 mice/group) for 5 days. A representative flow cytometry plot is shown. (b) Histograms report the mean values±S.D. of CD1a (top) and CD4 (bottom) in all mice analyzed. Data were analyzed with Mann–Whitney test with Bonferroni correction (**P<0.01)

Figure 4

Positive enrichment of HDACi-related pathways in Givinostat-treated PDX by GSEA. (a) Heat maps depict for each set of xenografts supervised analysis of differentially expressed probes (LFDR<0.05) comparing Givinostat versus vehicle; PD-TALL9 (left), PD-TALL8 (middle) and PD-TALL16 (right) mice treated with Givinostat or vehicle for 6 h. The number of differentially expressed genes are reported. (b) GSEA plots of one of the top enrichment sets (HELLER_HDAC_UP) for PD-TALL9 (left), PD-TALL8 (middle) and PD-TALL16 (right) are shown. (c) Heat map representation of the top 25 differentially expressed genes in PD-TALL9 (left), PD-TALL8 (middle) and PD-TALL16 (right). The columns show individual samples. Red and blue indicate higher and lower expression levels, respectively

Figure 5

Identification of genes differentially regulated in good compared with poor responders upon Givinostat treatment and IPA analysis. (a) Venn diagram showing the common response (293 genes) to Givinostat treatment in both good responders (PD-TALL8 and PD-TALL16) compared with the poor responder (PD-TALL9). The list of 293 common genes results from the intersection between the genes specifically modulated by Givinostat treatment in each good responder compared with the poor responder (Giv8 versus Giv9 and Giv16 versus Giv9); genes that at the basal level are already differently expressed were removed for each set (Vehicle 8 versus Vehicle 9 and Vehicle 16 versus Vehicle 9). (b) Top canonical pathways for the list of 291 genes that characterized the good and poor response to Givinostat using IPA analysis. Results are scored based on the negative base 10 logarithm of the P-value (bars). The different color of the bars represent the predicted activation (z-score) for each canonical pathway. Orange lines: ratio, calculated as the ratio between the number of genes found in a pathway and the total number of genes that constitute that specific canonical pathway

Figure 6

Good responders upregulated DNA repair genes compared with poor responders upon Givinostat treatment and increased DNA damage protein pH2AX. (a) RAD50, MLH1, NBN, CDC73, JAG1 and DLL1 expression analysis by qRT-PCR in good (PD-TALL8, PD-TALL16, PD-TALL43) and poor responders (PD-TALL9 and PD-TALL6) after 6 h of treatment with Givinostat in vivo. PD-TALL8 (n=2 mice), PD-TALL16 (n=5 mice), PD-TALL43 (N=4 mice), PD-TALL9 (n=4 mice), PD-TALL6 (n=3 mice). The 2^{−Delta CT} (Delta CT=CT gene–CT Beta2 microglobulin) was used as a read out of quantitative RT-PCR data. (b) Cells were recovered from the spleen of the xenografts and treated in vitro with Givinostat or vehicle for 6 h. pH2AX and total H2AX protein levels were then analyzed by western blotting