The TLK-ASF1 histone chaperone pathway plays a critical role in IL-1β-mediated AML progression

Abstract

Identifying and targeting microenvironment-driven pathways that are active across acute myeloid leukemia (AML) genetic subtypes should allow the development of more broadly effective therapies. The proinflammatory cytokine interleukin-1β (IL-1β) is abundant in the AML microenvironment and promotes leukemic growth. Through RNA-sequencing analysis, we identify that IL-1β-upregulated ASF1B (antisilencing function-1B), a histone chaperone, in AML progenitors compared with healthy progenitors. ASF1B, along with its paralogous protein ASF1A, recruits H3-H4 histones onto the replication fork during S-phase, a process regulated by Tousled-like kinase 1 and 2 (TLKs). Although ASF1s and TLKs are known to be overexpressed in multiple solid tumors and associated with poor prognosis, their functional roles in hematopoiesis and inflammation-driven leukemia remain unexplored. In this study, we identify that ASF1s and TLKs are overexpressed in multiple genetic subtypes of AML. We demonstrate that depletion of ASF1s significantly reduces leukemic cell growth in both in vitro and in vivo models using human cells. Using a murine model, we show that overexpression of ASF1B accelerates leukemia progression. Moreover, Asf1b or Tlk2 deletion delayed leukemia progression, whereas these proteins are dispensable for normal hematopoiesis. Through proteomics and phosphoproteomics analyses, we uncover that the TLK-ASF1 pathway promotes leukemogenesis by affecting the cell cycle and DNA damage pathways. Collectively, our findings identify the TLK1-ASF1 pathway as a novel mediator of inflammatory signaling and a promising therapeutic target for AML treatment across diverse genetic subtypes. Selective inhibition of this pathway offers potential opportunities to intervene effectively, address intratumoral heterogeneity, and ultimately improve clinical outcomes in AML.

Graphical abstract

Figure 1.

ASF1B is upregulated in AML progenitors upon IL-1β stimulation. (A) RNA-seq analysis was performed using purified CD34⁺ cells derived from the bone marrow of patients with AML (n = 3) and healthy donors (n = 3). Cells were cultured with 10 ng/mL IL-1β or vehicle control for 2 days. The heat map indicates log₂ counts per million (CPM) of differentially expressed genes in vehicle- or IL-1β–treated healthy and AML CD34⁺ bone marrow cells. (B) STRING-DB network analysis of genes upregulated in IL-1β–treated AML cells vs IL-1β–treated healthy cells. (C) TLDA analysis was performed on AML (n = 4) and healthy CD34⁺ (n = 4) bone marrow cells treated with or without 10 ng/mL IL-1β for 2 days. The expression of listed genes is shown as a fold difference upon IL-1β stimulation compared with respective vehicle controls in AML and healthy CD34⁺ bone marrow cells. (D) Schematic representation of the TLK-ASF1 pathway. (E) Immunoblot of TLKs and ASF1s protein expression in CD34⁺ bone marrow cells from healthy donors (n = 5) and patient-derived AML samples (n = 14) with indicated mutations. (F) Densitometry analysis of the immunoblot is shown and expressed as means ± standard error of the mean (SEM). Student t test was used for statistical analysis. ∗P < .05.

Figure 2.

Reduced ASF1 levels suppressed in vitro and in vivo AML growth and leukemia burden using human cells. (A) A schematic representation of a doxycycline-inducible shRNA model targeting ASF1A, ASF1B, or both ASF1A and ASF1B in MOLM-14 cells. (B) MOLM-14 cells were treated with 1 μg/mL doxycycline to induce knockdown for 6 days. The effect of the knockdown was measured by immunoblotting analysis. shRNA expressing cells were kept in doxycycline-containing media throughout the culture. (C) Cell viability of MOLM-14 cells expressing shASF1A, shASF1B, or both was measured by colorimetric (MTS) assays after 6 days of doxycycline-induced knockdown. (D) Cell growth over time of MOLM-14 cells expressing shASF1A, shASF1B, or both was indicated by cell counts. (E) Cell cycle analysis of MOLM-14 cells expressing shASF1A, shASF1B, or both using propidium iodide and flow cytometry after 3 days of doxycycline-induced knockdown. (F) Colony formation assay was assessed by plating MOLM-14 cells expressing shASF1A, shASF1B, or both in the methylcellulose-based medium following doxycycline-induced knockdown for 14 days. (G) MOLM-14 cells expressing shASF1A, shASF1B, or both were xenografted into sublethally irradiated NSG mice, which were randomly divided into 2 groups (n = 4-5 per group) with or without 625 mg/kg doxycycline chow. All the mice were euthanized 18 days after treatment. (H-I) The leukemic burden was determined by GFP and/or RFP positivity, human CD13/CD33, and CD15 positivity using flow cytometry. (J) Spleen weight and representative images of the spleens are shown from each group. Data are expressed as means ± SEM and the Student t tests were used for statistical analyses. ∗∗P < .01 and ∗∗∗P < .001.

Figure 3.

ASF1B potentiated IL-1–driven AML progression in a murine model of leukemia, and the absence of ASF1B attenuated the leukemia progression. (A) A schematic representation of a murine AML bone marrow transduction/transplantation model mimicking ASF1B overexpression. Lineage (Lin⁻)-depleted wild-type bone marrow cells were transduced with MSCV-IRES-mCherry or MSCV-IRES-ASF1B-mCherry and MSCV-IRES-MLL-ENL-GFP constructs. After 48 hours of transduction, 10 000 sorted mCherry⁺GFP⁺ cells and supporting cells were injected into lethally irradiated C57BL/6 recipients. After engraftment confirmation, recipient mice were treated with 0.25 μg IL-1β or vehicle and monitored daily. The recipient mice were euthanized when they appeared moribund or showed signs of sickness. (B) Kaplan-Meier survival curve of recipient mice. The log-rank test was used for statistical analysis. ∗P < .05 and ∗∗P < .01 (C) Immunophenotyping of Asf1b^+/+, Asf1b^+/−, and Asf1b^−/− mice representing different cell populations in the bone marrow. The distribution of stem and progenitor cells in the bone marrow of Asf1b^+/+, Asf1b^+/−, and Asf1b^−/− mice was measured by flow cytometry. (D) A schematic representation of a murine AML bone marrow transduction/transplantation model to study the effect of ASF1B deletion on leukemia progression. Lineage (Lin⁻)-depleted Asf1b^+/+, Asf1b^+/−, and Asf1b^−/− bone marrow cells were transduced with MSCV-IRES-MLL-ENL-GFP retrovirus. After 48 hours of transduction, 25 000 sorted Lin⁻GFP⁺ cells and supporting cells were injected into lethally irradiated C57BL/6 recipients. After engraftment confirmation, recipient mice were treated with 0.25 μg IL-1β or vehicle and monitored daily. (E) Kaplan-Meier survival curve of recipient mice. The log-rank test was used for statistical analysis. ∗∗P < .01.

Figure 4.

Reduced expression of TLKs suppresses in vitro and in vivo AML growth and leukemia burden using human cells. (A) A schematic representation of a doxycycline-inducible shRNA model targeting TLK1, TLK2, or both TLK1 and TLK2 in MOLM-14 cells. (B) MOLM-14 cells were treated with 1 μg/mL doxycycline for 6 days to induce knockdown. The effect of the knockdown was measured by immunoblotting analysis. (C) Cell viability of MOLM-14 cells expressing shTLK1, shTLK2, or both was measured by colorimetric (MTS) assay after 6 days of doxycycline-induced knockdown. (D) Cell growth over time of MOLM-14 cells expressing shTLK1, shTLK2, or both was indicated by cell counts. (E) Cell cycle analysis of MOLM-14 cells expressing shTLK1, shTLK2, or both using propidium iodide and flow cytometry after 6 days of doxycycline-induced knockdown. (F) Colony formation was assessed by plating cells in methylcellulose-based medium after doxycycline-induced knockdown for 14 days. Data are expressed as means ± SEM. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001.

Figure 5.

The absence of TLK2 attenuates leukemia burden and IL-1β–driven AML progression in a murine model of leukemia. (A) A schematic representation of a murine AML bone marrow transduction/transplantation model to study the effect of TLK2 deletion on leukemia progression. Lineage-depleted Tlk2^+/+, Tlk2^+/−, Tlk2^−/− bone marrow cells were transduced with MSCV-IRES-MLL-ENL-GFP retrovirus. After 48 hours of transduction, 15 000 sorted Lin⁻GFP⁺ cells and supporting cells were injected into lethally irradiated C57BL/6 recipients. After engraftment confirmation, recipient mice were treated with 0.25 μg IL-1β or vehicle and monitored daily. (B) Kaplan-Meier survival curve of recipient mice. The log-rank test was used for statistical analysis. (C) The percentage of leukemic cells in the peripheral blood over time was measured by flow cytometry. ∗P < .05, ∗∗P < .01, and ∗∗∗∗P < .0001.

Figure 6.

Global proteome and phosphoproteome alterations in AML cells upon ASF1 and TLK depletion. Global proteomics and phosphoproteomics analysis of AML cell line MOLM-14 was performed upon knockdown of ASF1s or TLKs as described in supplemental Figure 6A and “Methods.” (A) STRING-DB Gene Ontology analysis for the biological process for proteins that were significantly downregulated for protein levels or their phosphorylation upon ASF1A/B knockdown (left) compared with nontargeting controls (shNT). Analysis of variance (ANOVA) t test, false discovery rate (FDR) <0.05. Additionally, the top 50 downregulated phosphorylation sites for the proteins associated with cell cycle (middle, GO:0007049) and DNA repair (right, GO:0006281) Gene Ontology terms are shown. Scale bar represents normalized TMT intensity using medium centering approach. If multiple phosphorylation sites of the same protein were observed, only the most downregulated phosphorylation sites were shown in the heat maps. (B) The x-axis corresponds to the normalized TMT intensity with medium centering approach of global or phosphorylated protein of all proteins that were significantly altered in shASF1A/B cells (ANOVA t test, FDR < 0.05), whereas the y-axis shows their normalized TMT intensity in shASF1A, shASF1B, or shNT cells. The correlation coefficients (r) are shown for shASF1A/B cells with shASF1A (red), shASF1B (blue), and shNT (gray) cells within cell cycle and DNA repair Gene Ontology terms. (C) Venn diagram showing the overlap of proteins with reduced phosphorylation in MOLM-14 cells with ASF1A/B or TLK1/2 knockdown. (D) STRING-DB Gene Ontology analysis for the biological process for the 446 proteins with reduced phosphorylation in MOLM-14 cells with both ASF1A/B or TLK1/2 knockdown. (E) Representative phosphorylated DNA damage proteins upon ASF1 and TLK knockdown. (F) Immunoblot of DNA damage and cell cycle–related proteins upon ASF1 and TLK knockdown. TMT, tandem mass tags.