Cytosolic DNA sensing combined with differentiation therapy induces irreversible differentiation and cell growth arrest in myeloid leukemia cells

doi:10.21203/rs.3.rs-2047508/v1

Download PDF

Research Article

Cytosolic DNA sensing combined with differentiation therapy induces irreversible differentiation and cell growth arrest in myeloid leukemia cells

https://doi.org/10.21203/rs.3.rs-2047508/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

Accumulating evidence indicates that various oncogenic mutations interfere with normal myeloid differentiation of leukemogenic cells during the early process of acute myeloid leukemia (AML) development. Differentiation therapy is an ideal therapeutic strategy capable of terminating leukemic expansion by reactivating the differentiation potential; however, the plasticity and instability of leukemia cells counteract the establishment of curative treatment for AML. Thus, the viable strategy for irreversible differentiation is urgently required. Based on our previous observation that autophagy inhibitor treatment induces the accumulation of cytosolic DNA and activation of cytosolic DNA-sensor signaling selectively in leukemia cells, we herein examined the synergistic effect of cytosolic DNA-sensor signaling activation with conventional differentiation therapy on AML. The combined treatment with autophagy inhibitors and conventional differentiation therapy succeeded in inducing irreversible differentiation in AML cells. Mechanistically, cytosolic DNA was sensed by absent in melanoma 2 (AIM2), a cytosolic DNA sensor. Activation of the AIM2 inflammasome resulted in the accumulation of p21 through the inhibition of its proteasomal degradation, thereby facilitating the myeloid differentiation. Moreover, p21 shRNA treatment abrogated the combined treatment-induced irreversible differentiation of leukemia cells. Importantly, short-term treatment with the combined therapy was sufficient to induce irreversible differentiation and dramatically reduced the total leukemia cell counts and proportion of blast cells in the spleens of AML mice. Collectively, these findings indicate that the activation of cytosolic DNA-sensor signaling in combination with conventional differentiation therapy can be an effective approach to drive the irreversible differentiation of leukemia cells.

AML

Differentiation therapy

cytosolic DNA

Acute myeloid leukemia (AML) is a heterogenous hematopoietic malignancy caused by various oncogenic mutations. However, accumulating evidence suggests that many genetic and epigenetic alterations commonly block normal myeloid differentiation of immature states of leukemia cells, thereby leading to their uncontrollable proliferation (1). Thus, the therapeutic treatment of pharmacological agents, which can promote the differentiation of immature leukemia cells, have been investigated and is termed as ‘differentiation therapy’ (1). A representative agent, all-trans retinoic acid (ATRA), succeeded in ameliorating the prognosis of acute promyelocytic leukemia (APL), which is caused by a chromosomal translocation that results in a gene fusion encoding an oncoprotein, PML-RARA (2). At secondary relapse, however, some cases presented with resistance to ATRA or mature leukemia cells whose leukemogenic property had been regained through de-differentiation, especially in patients treated with low-dose consolidation (3, 4). Moreover, ATRA can promote differentiation in some types of non-APL AML cells by activating the normal retinoic acid receptor. However, ATRA is generally ineffective in patients with non-APL AML (5–8). Meanwhile, leukemia cells progress towards matured states in patients treated with molecular targeted therapies against several driver mutations, including mutant isocitrate dehydrogenase (IDH)1, IDH2, and FLT3, emphasizing that differentiation therapy is also clinically applicable to non-APL (1). Therefore, effective interventions enabling irreversible differentiation in leukemia cells are urgently needed.

Cytosolic double-stranded (ds)DNA is released from nuclei with a compromised integrity and can be recognized by various sensors as a damage-associated molecular pattern. Cytosolic DNA-sensors are functionally divided into those concerning type Ⅰ IFN production and those concerning inflammasome activation (9). In the former group, a well-studied DNA-sensor is cyclic GMP-AMP (cGAMP) synthase (cGAS), which produces cGAMP after sensing dsDNA in the cytoplasm (10, 11). Then, cGAMP is recognized by a central adaptor molecule, stimulator of IFN genes (STING), leading to type Ⅰ IFN production (12). On the other hand, a representative inflammasome-activating DNA-sensor, AIM2, can induce IL-1β maturation and pyroptosis, a type of immunogenic cell death, via enzymatic digestion by caspase-1 (13–16). Cytosolic DNA accumulation is preferentially observed in various types of malignant cells and can be ascribed to DNA release from multiple origins (17). However, the biological effects of cytosolic DNA in cancer remain elusive.

We previously observed that myeloid leukemia cells constitutively leak dsDNA into the cytoplasm, probably due to uncontrolled cell cycle progression and subsequent genome instability (18). A detailed analysis of cytosolic DNA dynamics revealed that cytosolic DNA can be degraded in leukemia cells through autophagy. Furthermore, inhibition of autophagosome formation, the early autophagy event, but not autolysosome formation, the late event, results in cytosolic dsDNA accumulation, activating cytosolic DNA-sensor signaling pathways. Thus, autophagy inhibition-induced accumulation of cytosolic DNA can be an effective treatment approach against AML. Here, we examined the potential synergistic effect of cytosolic DNA-sensor signaling activation and conventional differentiation therapy on AML. Of interest, short-term treatment with the combined therapy with autophagy inhibitors and ATRA sufficiently induced irreversible myeloid differentiation in AML cells by activating the cytosolic DNA-sensor, AIM2.

Mice. Specific pathogen-free 6- to 8-week-old male BALB/c mice were purchased from Charles River Japan. All mice were kept under specific pathogen-free conditions, housed (five per cage) in a temperature (21–25°C), and maintained on a 12 h light/dark cycle (08:00 to 20:00 lights on). Mice were provided standard food and water ad libitum. All the animal experiments complied with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University.

Antibodies (Abs). The following rat anti-mouse monoclonal (m)Abs; anti-CD11b (M1/70, TONBO Biosciences) and anti-CD19 (1D3, TONBO Biosciences), and mouse or rabbit anti-human mAbs; anti-CD38 (HB7, TONBO Biosciences), anti-p21 (12D1, Cell Signaling), anti-AIM2 (3B10, BioLegend), anti-SKP2 (EPR3305(2), abcam), and anti-USP10 (D7A5, Cell Signaling) were used. Isotype-matched control IgGs for individual rat and mouse mAb were purchased from BD Biosciences. PE-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific) was used as the secondary Ab for the flowcytometry analysis.

Culture of cell lines and cell repopulation assay. The KG-1(19) (JCRB0065) cell line was obtained from JCRB Cell Bank. The human AML cell lines, HL-60, THP-1, KG-1, MOLM-13, and MOLM-14 as well as the human CML cell line, K562, were cultured in RPMI-1640 medium supplemented with 1 % FBS. In the repopulation assay, cells were washed twice with fresh culture medium at the indicated time periods of drug treatment. Then, viable cells were counted using trypan blue staining method and replated in 96-well plates (10⁴ viable cells/well). Cell proliferation was determined using a cell counting kit-8 (DOJINDO) at the indicated time points until confluency was reached.

Establishment of knock-down (KD) cell lines. STING and AIM2 KD cells under the background of HL-60 and THP-1 cell lines have been previously established.(18) AIM2 KD MOLM-14 cell lines were newly established by infection with MISSION lentivirus carrying pLKO.1 puro with shRNA constructs (AIM2-1: TRCN0000107503, AIM2-2: TRCN0000107504), which were purchased from Sigma-Aldrich. To establish p21 KD cells, HL-60 cells were infected with MISSION lentivirus carrying pLKO.1 puro with shRNA constructs (p21-1: TRCN0000040123, p21-2: TRCN0000287021). As a control, each cell line was infected with MISSION lentivirus carrying pLKO.1 puro with non-mammalian shRNA control constructs. Stably transfected cells were selected in culture medium supplemented with 1 µg/ml puromycin for more than two weeks.

Retroviral preparation. The MSCV-MLL-AF9-ires-GFP vector was a gift from Akihiko Yokoyama, National Cancer Center, Tsuruoka Metabolomics Laboratory, Japan. Retroviral packaging cells (Phoenix 293T) were transiently transfected with the MSCV-MLL-AF9-ires-GFP plasmid using jetPRIME transfection reagent (Polyplus-transfection) to produce the retrovirus carrying MSCV-MLL-AF9-ires-GFP in the culture supernatant, which was subsequently used to infect lineage⁻c-kit⁺sca-1⁺ (LKS⁺) cells isolated from mouse bone marrow (BM).

Generation of mouse AML model. LKS⁺ cells purified from the BM of BALB/c mice were cultured in serum-free S-Clone SF-03 medium (Sanko Junyaku) supplemented with 1% BSA, 100 ng/ml stem cell factor (PeproTech), 100 ng/ml thrombopoietin, 25 ng/ml fms-like tyrosine kinase-3 ligand (PeproTech), 10 ng/ml IL-6 (PeproTech), and 10 ng/ml IL-3 (PeproTech) for 24 h. Cultured LKS⁺ cells were infected with the retrovirus carrying MSCV-MLL-AF9-ires-GFP using a ViroMag R/L kit (OZ Bioscience) to obtain leukemia initiating cells. The resultant cells (50–100 GFP⁺ cells included in 30,000 KLS⁺ cells) were intravenously transplanted into 5 Gy X-irradiated recipient BALB/c mice along with 3 × 10⁵ normal BM cells in a 200 µl volume. Total BM cells were harvested from primary AML mice and stored in liquid nitrogen. A frozen stock of primary AML cells was cultured in semi-solid methylcellulose-based medium (Methocult GF M3534, STEMCELL technologies). To prepare the mouse AML model, 2 × 10⁵ colony forming cells were intravenously transplanted into sub-lethally irradiated secondary recipient mice along with 1 × 10⁶ normal BM cells.

In vivodrug administration. ATRA (Sigma-Aldrich) and MRT-68921 (MRT) (Sigma-Aldrich) were dissolved in DMSO and distilled water to make a 2.5 and 20 mg/ml stock solution, respectively. AML-bearing mice were intraperitoneally injected with ATRA (5 mg/kg, diluted in PBS containing 5% polyethylene glycol 400 (Sigma-Aldrich) and 5% Tween 80 (Sigma-Aldrich)) or MRT (20 mg/kg, diluted in PBS according to the schedule) (Fig. 5a and e).

Extraction and measurement of cytosolic dsDNA. The cytoplasmic fraction of leukemia cells was extracted using a cell fractionation kit (Abcam). The concentration of dsDNA was specifically measured with Qubit 4 (Thermo Fisher Scientific) using a Qubit 1X dsDNA HS assay kit (Thermo Fisher Scientific).

NBT reduction assay. One nitro blue tetrazolium (NBT) tablet (Sigma-Aldrich) was dissolved in 10 ml culture medium and used as the working solution. Cells were incubated with 10 µg/ml PMA in the NBT working solution for 20 min at 37°C. Then, their cytospin-prepared slides were fixed with methanol and counterstained with 0.1 % safranin O for 30 seconds.

RNA extraction, cDNA synthesis, and qRT-PCR. Total RNAs were extracted from cells using an RNeasy Mini Kit (QIAGEN) and then reverse-transcribed using the SuperScript Ⅳ VILO (Thermo Fisher Scientific). qRT-PCR was performed on StepOne Real-time PCR system (Thermo Fisher Scientific) using the Luna Universal qPCR Master Mix (New England BioLabs). The following specific primer sets were used: human CXCL3 gene (sense: 5’-ACC GAA GTC ATA GCC ACA CTC-3’; antisense: 5’-AGT TGG TGC TCC CCT TGT T-3’), human GAPDH gene (sense: 5’-GCC AAA AGG GTC ATC TC-3’; antisense: 5’-TGA GTC CTT CCA CGA TAC CA-3’), human NF-κB2 gene (sense: 5’-CTC GAA TGG ACA AGA CAG CA-3’; antisense: 5’-TTA CAG GCC GCT CAA TCT TC-3’), human P21 gene (sense: 5’-AGG GGA CAG CAG AGG AAG AC-3’; antisense: 5’-GGC GTT TGG AGT GGT AGA AA-3’), human RRM2 gene (sense: 5’-CTG GCT CAA GAA ACG AGG AC-3’; antisense: 5’-GTT TGA ACA TCA GGC AAG CA-3’), human TNF-α gene (sense: 5’-GGC GTG GAG CTG AGA GAT AA-3’; antisense: 5’-GAT GGC AGA GAG GAG GTT GA-3’), human TYMS gene (sense: 5’-TCC CGA GAC TTT TTG GAC AG-3’; antisense: 5’-TCA GGG TTG GTT TTG ATG GT-3’). The relative expression of each gene was analyzed by the ΔΔCt method using the Ct value of the GAPDH gene.

Flow cytometry. Intracellular p21, AIM2, SKP2, and USP10 were stained with each protein-specific mAb using a Foxp3/Transcription Factor Buffer Set (eBioscience). For cell cycle analysis, cells were stained with Vybrant DyeCycle Green (Thermo Fisher Scientific). The expression of each molecule was determined using a FACSCantoII (BD Biosciences) and analyzed with FlowJo software (Tree Star).

IP and western blot analysis for the detection of ubiquitylated p21. Cells were lysed in CelLytic M (Sigma) containing % Proteoguard (Clontech) for 15 min at 4°C. The supernatants were harvested after centrifugation at 15,000 g for 15 min and subjected to immunoprecipitation (IP) to isolate the p21 protein using a rabbit anti-human p21 mAb (E2R7A, Cell Signaling) and a Dynabeads Protein G IP kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The resultant IP samples were subjected to SDS-PAGE and blotted onto nitrocellulose membranes. The blots were probed with rabbit anti-p21 mAb (E2R7A) or mouse anti-ubiquitin mAb (UBCJ2, Enzo Life Sciences) and reacted with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch). The immune complexes were visualized using ImmunoStar LD (FUJIFILM Wako Chemicals) and detected with Imager 680 (Cytiva). Signal intensities were analyzed by ImageJ software. As a negative control, we confirmed that the GAPDH protein was hardly detected in any of the IP samples.

Microarray analysis. Total RNA was extracted and its quality was checked using an Agilent Bioanalyzer (Agilent Technologies). The cDNA was synthesized using a GeneChip Whole Transcript Amplification kit (Thermo Fisher Scientific). The sense cDNA was then fragmented and biotin-labeled with terminal deoxynucleotidyl transferase using a GeneChip WT Terminal Labeling kit (Thermo Fisher Scientific). Labeled DNA (5.5 µg) was hybridized to the Affymetrix GeneChip Array (Clariom S Assay human, Thermo Fisher Scientific) at 45 ℃ for 16 h. Slides were washed, stained on a GeneChip Fluidics Station 450 (Thermo Fisher Scientific), and scanned with a GCS3000 Scanner (Thermo Fisher Scientific). The probe cell intensity data computation and CEL file generation were performed using Affymetrix GeneChip Command Console software (Thermo Fisher Scientific). The obtained data were normalized and filtered using Affymetrix Power Tools (Thermo Fisher Scientific), and then subjected to gene set enrichment analysis (GSEA). Differentially expressed genes (Fold change ≥ 3) were subjected to GO functional analysis (Data source category: Biological process) using gProfiler. GSEA was executed through GenePattern server. As the metric for ranking genes, the log2_ratio_of_means was exploited, and the gene set size was set to 15–500. Hallmark gene sets were used in this study.

Statistical analysis. We did not use specific sample size calculation methods. Any technically validated data were not excluded. Data were analyzed statistically using GraphPad Prism software (Ver. 6; La Jolla, CA, USA) using the methods indicated in the legend of each figure. Two-sided Student’s t-test and one-way ANOVA followed by Tukey-Kramer post-hoc test were used to compare the data among two and more than two groups, respectively. The log-rank test was used to evaluate the survival curve data. Statistical significance was set at p < 0.05.

Combined treatment with autophagosome formation inhibitors and ATRA induces irreversible differentiation in myeloid leukemia cells. We previously reported that Atg5-specific siRNA treatment and autophagosome formation inhibitors, MRT and SBI-0206965 (SBI), but not an autolysosome inhibitor, hydroxychloroquine, induced accumulation of cytosolic DNA fragments preferentially in leukemia cells (18). This prompted us to examine the effects of MRT on ATRA-induced cytotoxicity in HL-60 cells, which are myeloid leukemia cells without the PML-RARA mutation (20). Indeed, MRT synergistically enhanced ATRA-induced cytotoxicity (Fig. 1a). Moreover, combined treatment with ATRA and MRT (ATRA + MRT), but not monotherapy with either ATRA or MRT, induced nuclear indentation or segmentation in HL-60 cells, which is characteristic of myeloid differentiation (Fig. 1b). ATRA + MRT rapidly induced the expression of an early myeloid cell differentiation marker, CD38, to the same extent as that induced by ATRA alone; however, only ATRA + MRT induced decreased CD38 expression in CD11b^high cells (Fig. 1c), a late differentiation phenotype (21). Furthermore, 24 h- and 48 h-pre-exposure to ATRA + MRT irreversibly prevented cell proliferation even under the drug-free conditions (subsequently referred to as irreversible cell growth arrest), whereas pre-exposure to either ATRA or MRT failed to do so (Fig. 1d and Supplementary Figure 1). Consistently, HL-60 cells treated with ATRA + MRT for 24 h continuously progressed toward a late differentiation phenotype and eventually died under the same condition, whereas cells pre-treated with ATRA alone only showed transiently enhanced CD38 expression and finally exhibited a CD38^lowCD11b^medium phenotype, which was similar to that of untreated cells (Fig. 1e). Additionally, ATRA + SBI induced myeloid differentiation to the same extent as ATRA + MRT, as evidenced by increased NBT staining, and eventually caused irreversible cell growth arrest (Supplementary Fig. 2). Similar morphological changes and cell growth arrest were observed when other myeloid leukemia cell lines (THP-1, K562, and KG-1) were treated with ATRA + MRT (Supplementary Fig. 3). Thus, the results suggest that autophagosome formation inhibitors in combination with ATRA can induce irreversible myeloid differentiation.

The AIM2 inflammasome drives irreversible differentiation in leukemia cells. ATRA + MRT, but not ATRA, markedly increased the content of cytosolic dsDNA in HL-60 cells (Fig. 1f), confirming our previous observation of cytosolic dsDNA accumulation by autophagosome formation inhibitors (18). Since the AIM2 and STING pathways are major cytosolic DNA-sensor signaling pathways (9), each of the molecules was silenced with the use of shRNAs in HL-60 cells to address their contribution. AIM2-, but not STING-specific shRNA transduction, rescued the ATRA + MRT-mediated irreversible cell proliferation blockage (Fig. 1g) and enhanced CD11b expression (Fig. 1h). Among two AIM2 KD cell lines, AIM-2-2 exhibiting more efficient recovery of cell growth was mainly subjected to the subsequent experiments as shAIM2. Cytosolic DNA sensing induces the AIM2 inflammasome, activating an inflammatory caspase, caspase-1, and thereby exerting biological functions such as IL-1β activation and pyroptotic cell death (16). Hence, we next explored the roles of caspase-1 and IL-1β in this process. The increased expression of CD11b induced by ATRA + MRT was reduced by treatment with the caspase-1 inhibitor, Z-WEHD, but not with an IL-1β blocking antibody (Fig. 1i and Supplemental Fig. 4). Thus, the AIM2-caspase-1 pathway drives ATRA + MRT-induced irreversible differentiation in myeloid leukemia cells independent of IL-1β autocrine signals.

The involvement of p21 proteasomal degradation inhibition in AIM2-mediated myeloid differentiation of leukemia cells. Consistent with the functional observations, transcriptomic analysis revealed augmented expression of leukocyte activation-related genes in differentiated HL-60 cells treated with ATRA + MRT, whereas the expression of cell cycle-related genes was decreased (Fig. 2a and b). qRT-PCR confirmed that the changes in the expression of representative genes among each gene set were consistent with the results of the transcriptomic analysis (Supplemental Fig. 5). Additionally, AIM2 KD significantly modulated the changes in mRNA levels of these differentially expressed genes (Supplemental Fig. 6), except for those of the p21 gene (Fig. 2c), the gene presumed to be essential for ATRA-mediated myeloid differentiation in APL cells (22). In fact, combined treatment induced a greater enhancement of p21 protein levels than ATRA monotherapy (Fig. 2d), which, however, was significantly inhibited in AIM2 KD (Fig. 2e). This inhibition of p21protein but not mRNA expression was similarly observed in another AIM2 KD cell line (data not shown). Likewise, AIM2 shRNA treatment reduced the ATRA + MRT-mediated up-regulation of CD11b and p21 protein expression in THP-1 cells, another human leukemia cell line (Supplemental Fig. 7). Furthermore, ATRA + MRT treatment induced G1 arrest in HL-60 cells, and this G1 arrest was released by AIM2 KD (Fig. 2f). These findings suggest that the AIM2 inflammasome can control p21 protein levels at a post-translational level, thereby regulating cell cycle progression. Indeed, the AIM2 KD-induced decrease in p21 protein levels was abrogated by a proteasome inhibitor, MG-132 (Fig. 3a). Thus, the AIM2 inflammasome can regulate p21 proteasomal degradation, which is presumed to be tightly controlled by SCF-SKP2 E3 ligase complex-mediated ubiquitylation (23). SKP2 is itself regulated by ubiquitylation and can be stabilized by a deubiquitinase, USP10 (24), a potential substrate of caspase-1 (25). ATRA + MRT treatment reduced both USP10 and SKP2 protein levels as well as the ratio of polyubiquitylated p21, while these decreases were reduced by AIM2 KD (Fig. 3b–d). Furthermore, a USP10 inhibitor, spautin-1, markedly attenuated the AIM2 KD-mediated decrease in p21 protein levels (Fig. 3e). Similarly, p21 KD (Supplemental Fig. 8) attenuated the ATRA + MRT-induced enhancement of CD11b expression (Fig. 3f) and irreversible cell growth arrest (Fig. 3g). Together, the results demonstrated that ATRA + MRT treatment can activate the AIM2 inflammasome-caspase-1 pathway to inhibit p21 proteasomal degradation, promoting myeloid differentiation and cell growth arrest in leukemia cells.

Autophagy inhibitors together with an FLT3 inhibitor synergistically induces irreversible differentiation in leukemia cells with the FLT3 mutation. In a clinical study, FLT3 inhibitors effectively reduced the numbers of leukemia blast cells in peripheral blood, but not those in the BM, where the cells were relatively resistant to these drugs (26). This drug resistance can be partially ascribed to stromal cell-derived FGF2 (27). Consistently, FGF2 blunted the cytotoxicity and retardation of cell proliferation induced by the FLT3 inhibitor, quizartinib, after drug removal in MOLM-14, a human AML cell line carrying an FLT3 internal tandem duplication mutation (FLT3-ITD) (Fig. 4a and b). However, combined incubation with quizartinib and MRT blocked cell repopulation in a dose-dependent manner even in the presence of FGF2 (Fig. 4c). The same treatment further induced up-regulation of CD11b expression (Fig. 4d) and emergence of NBT⁺ cells with segmented nuclei (Supplemental Fig. 9). Moreover, AIM2 KD inhibited the increase in p21 protein levels, appearance of cells with segmented nuclei, and cell growth arrest in MOLM-14 cells treated with quizartinib and MRT (Supplemental Fig. 10 and Fig. 4e). Thus, the autophagosome formation inhibitors can also synergize with a molecular-targeted drug, FLT3 inhibitor, to induce irreversible differentiation in leukemia cells with FLT3-ITD via the AIM2 inflammasome-caspase-1 pathway.

Therapeutic effects of short-term treatment with ATRA and MRT in the AML mouse model. As MLL-AF9-positive AML cells are relatively sensitive to ATRA-mediated myeloid differentiation (28), we next explored the optimal drug conditions against human AML cell line carrying MLL-AF9. A 24 h pre-exposure to ATRA + MRT was sufficient to induce irreversible cell growth arrest, irrespective of ATRA pretreatment (Supplemental Fig. 11a). Interestingly, cell proliferation after drug removal was suppressed even by short-term (6 h) treatment with ATRA + MRT following ATRA pretreatment (Supplemental Fig. 11b). These in vitro data incited us to conduct a preclinical study using MLL-AF9-positive AML-bearing mice to test the therapeutic effects of ATRA + MRT treatment (Fig. 5a). Combined treatment failed to cure AML, but significantly improved the survival rate (Fig. 5b), with the combined treatment group having a greater reduction in total WBCs and GFP⁺ leukemia cells in peripheral blood than the non-treatment or ATRA monotherapy groups (Fig. 5c and d). As the spleen (SP) and BM are major organs where leukemia cells propagate from blast cells, we examined the effects of combined treatment on cell composition in these organs (Fig. 5e). ATRA + MRT reduced total leukemia cells and the proportion of c-kit⁺ blast cells in the SP (Fig. 5f and g), but not in the BM (Fig. 5h and i). These negligible effects on BM may account for the failure to cure AML-bearing mice.

The G1 cell cycle stage is critical for determining whether hematopoietic cells will differentiate or proliferate. Thus, the role of the Cdk inhibitors, p21 and p27, in hematopoietic differentiation has been widely studied because of their regulatory function in the G1-S transition (29). Indeed, numerous studies have reported the crucial contribution of p21 to myeloid cell differentiation, including ATRA-induced APL cell differentiation (22, 30). Moreover, Goswami et al. (31) recently reported that the pharmacological activation of protein phosphatase 2A enforces p21-dependent terminal differentiation in various types of myeloid leukemia cells. Furthermore, p21 can directly interact with myeloid differentiation-related transcription factors, such as C/EBPα (32) and STAT3 (33), to modulate their molecular function reciprocally. Thus, we propose that AIM2 inflammasome-mediated accumulation of p21 can turn differentiation therapy into an irreversible process in leukemia cells.

Furthermore, we observed that AIM2 inflammasome activation by combined treatment with ATRA and MRT crucially reduced the expression of USP10, which can stabilize SKP2 (24), an essential modulator of p21 under cell cycle progression (23). Agard et al. (25) comprehensively investigated the substrates for inflammatory caspases and identified many proteins that can be cleaved by caspase-1, including USP10. Thus, caspase-1 can modify the molecular function of numerous proteins, including well-known substrates such as IL-1β and gasdermin D, through enzymatic cleavage upon inflammasome activation. Furthermore, Chadha et al. (34) recently reported that caspase-1 can directly degrade the ubiquitin ligase scaffold proteins, cullin-1 and cullin-5, thereby depressing the ubiquitylation activity of the RING E3 ligase complex. Likewise, caspase-1 may be able to directly degrade USP10 through enzymatic cleavage.

Here, combined treatment with ATRA and MRT induced a significant increase in the protein and mRNA expression of p21; however, the molecular mechanism whereby p21 is transcriptionally regulated remains elusive. The transcription of p21 appears to be controlled by both p53-dependent and -independent mechanisms (35). A certain p53-independent pathway may enhance p21 transcription in the HL-60 cell line, which is a p53-null cell line. Accumulating studies have unraveled the contribution of several transcription factors, such as Sp1, Sp3, AP2, STATs, C/EBPα, and C/EBPβ, to the p53-independent p21 transcriptional regulation (35); however, most of them are also involved in the differentiation of hematopoietic cells. Thus, the initial molecular event triggering p21 transcription during myeloid differentiation remains unclear.

Various driver mutations in AML directly interfere with myeloid differentiation processes. For instance, IDH1/2 mutations prevent α-ketoglutarate production and, instead, induce the accumulation of the oncometabolite, D-2-hydroxyglutarate (36), thereby disrupting the epigenetic regulation of normal hematopoietic differentiation by inactivating α-ketoglutarate-dependent enzymes, including the TET family of 5-methlycytosine hydroxylases and the jumonji-domain-containing group of histone lysine demethylases. Thus, inhibitors of mutant IDH1/2 can effectively prevent leukemogenic proliferation along with the promotion of myeloid differentiation (37). Moreover, the most frequently mutated gene in AML is FLT3, which is classified into two distinct alterations, either FLT3-ITD or point mutations in the tyrosine kinase domain. Radomska et al. (38) recently reported that FLT3 activation by its genetic mutation inhibits C/EBPα-induced myeloid differentiation through ERK1/2-mediated phosphorylation, and that FLT3 inhibitors can promote differentiation in leukemia cells with FLT3 mutations. Here, we found that MRT treatment can synergize with the FLT3 inhibitor, quizartinib, to induce the irreversible differentiation of FLT3-ITD⁺ AML cells, similarly to what was observed in ATRA-mediated differentiation. Collectively, the evidence suggests that the inhibition of autophagosome formation in combination with various types of anti-leukemia drugs capable of reactivating AML blast differentiation can drive irreversible differentiation.

Achieving sufficient therapeutic doses of anti-cancer agents inside the BM by systemic administration is arguably difficult (39). Indeed, we observed that in vivo administration of ATRA and MRT could reduce the total counts of leukemia cells and the proportion of c-kit⁺ blast cells in the SP. In contrast, the treatment was less effective in the BM, suggesting that the drug may have failed to reach a sufficient concentration in the BM. Many studies have developed novel strategies for drug delivery to the BM (39). Thus, we believe that further improvements in the administration of these drugs and the engineering of pharmacological devices with optimized drug delivery to the BM will pave the way for completing differentiation therapy and overcoming AML.

Myeloid differentiation is arrested by various oncogenic mutations in the early stages of malignant transformation, driving the uncontrollable proliferation of blast cells in AML. Thus, differentiation therapy is an ideal strategy for irreversibly blocking leukemic expansion. However, leukemia cells ongoing differentiation often reacquire leukemogenic properties through de-differentiation after the cessation of drug treatment. Here, we succeeded in inducing irreversible leukemia cell differentiation by activating cytosolic DNA-sensor signaling pathway in combination with conventional differentiation therapy. Mechanistically, blocking autophagic clearance resulted in the accumulation of cytosolic dsDNA fragments, activating the AIM2 inflammasome-caspase-1 pathway and consequently inhibiting p21 proteasomal degradation, which promoted myeloid differentiation and cell growth arrest in leukemia cells. Thus, this novel strategy can be an effective approach to improve the conventional differentiation therapy against AML.

Ab: Antibody, AIM2: absent in melanoma 2, AML: acute myeloid leukemia, APL: acute promyelocytic leukemia, ATRA: all-trans retinoic acid, BM: bone marrow, cGAMP: cyclic GMP-AMP, cGAS: cyclic GMP-AMP synthase, ds: double-stranded, FLT3-ITD: FLT3 internal tandem duplication mutation, GSEA: gene set enrichment analysis, IDH: isocitrate dehydrogenase, IP: immunoprecipitation, KD: knock-down, LKS⁺: lineage^-c-kit⁺sca-1⁺, m: monoclonal, MRT: MRT-68921, NBT: nitro blue tetrazolium, SBI: SBI-0206965, SP: spleen, STING: stimulator of IFN genes,

Ethics approval and consent to participate

Animal studies were approved by the Committee of Laboratory Animals of Kanazawa University (AP-214222). All the animal experiments complied with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University.

Consent for publication

Not applicable.

Availability of data and materials

Raw and processed microarray data are available at Gene Expression Omnibus (GEO) under accession number GSE193056 (GEO reviewer link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193056).

Competing Interest

The authors declare no competing financial interests.

Funding

This work was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 20K08749.

Authors Contribution

T.B. and U.T. designed, performed, and analyzed the experiments in their entirety. A.H., N.M., and Y.J. supervised the entire study. T.B. prepared the manuscript.

Acknowledgements

We thank the professional English-editing service (Editage).

Madan V, Koeffler HP. Differentiation therapy of myeloid leukemia: four decades of development. Haematologica. 2021;106(1):26-38.
Cicconi L, Lo-Coco F. Current management of newly diagnosed acute promyelocytic leukemia. Annals of oncology : official journal of the European Society for Medical Oncology. 2016;27(8):1474-81.
Martino OD, Welch JS. Retinoic Acid Receptors in Acute Myeloid Leukemia Therapy. Cancers. 2019;11(12).
McKenzie MD, Ghisi M, Oxley EP, Ngo S, Cimmino L, Esnault C, et al. Interconversion between Tumorigenic and Differentiated States in Acute Myeloid Leukemia. Cell stem cell. 2019;25(2):258-72 e9.
Belhabri A, Thomas X, Wattel E, Chelghoum Y, Anglaret B, Vekhoff A, et al. All trans retinoic acid in combination with intermediate-dose cytarabine and idarubicin in patients with relapsed or refractory non promyelocytic acute myeloid leukemia: a phase II randomized trial. The hematology journal : the official journal of the European Haematology Association. 2002;3(1):49-55.
Burnett AK, Hills RK, Green C, Jenkinson S, Koo K, Patel Y, et al. The impact on outcome of the addition of all-trans retinoic acid to intensive chemotherapy in younger patients with nonacute promyelocytic acute myeloid leukemia: overall results and results in genotypic subgroups defined by mutations in NPM1, FLT3, and CEBPA. Blood. 2010;115(5):948-56.
Estey EH, Thall PF, Pierce S, Cortes J, Beran M, Kantarjian H, et al. Randomized phase II study of fludarabine + cytosine arabinoside + idarubicin +/- all-trans retinoic acid +/- granulocyte colony-stimulating factor in poor prognosis newly diagnosed acute myeloid leukemia and myelodysplastic syndrome. Blood. 1999;93(8):2478-84.
Milligan DW, Wheatley K, Littlewood T, Craig JI, Burnett AK, Group NHOCS. Fludarabine and cytosine are less effective than standard ADE chemotherapy in high-risk acute myeloid leukemia, and addition of G-CSF and ATRA are not beneficial: results of the MRC AML-HR randomized trial. Blood. 2006;107(12):4614-22.
Briard B, Place DE, Kanneganti TD. DNA Sensing in the Innate Immune Response. Physiology. 2020;35(2):112-24.
Ablasser A, Goldeck M, Cavlar T, Deimling T, Witte G, Rohl I, et al. cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING. Nature. 2013;498(7454):380-4.
Sun L, Wu J, Du F, Chen X, Chen ZJ. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 2013;339(6121):786-91.
Wu J, Sun L, Chen X, Du F, Shi H, Chen C, et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science. 2013;339(6121):826-30.
Burckstummer T, Baumann C, Bluml S, Dixit E, Durnberger G, Jahn H, et al. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nature immunology. 2009;10(3):266-72.
Fernandes-Alnemri T, Yu JW, Datta P, Wu J, Alnemri ES. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature. 2009;458(7237):509-13.
Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G, Caffrey DR, et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature. 2009;458(7237):514-8.
Schroder K, Muruve DA, Tschopp J. Innate immunity: cytoplasmic DNA sensing by the AIM2 inflammasome. Current biology : CB. 2009;19(6):R262-5.
Kwon J, Bakhoum SF. The Cytosolic DNA-Sensing cGAS-STING Pathway in Cancer. Cancer discovery. 2020;10(1):26-39.
Baba T, Yoshida T, Tanabe Y, Nishimura T, Morishita S, Gotoh N, et al. Cytoplasmic DNA accumulation preferentially triggers cell death of myeloid leukemia cells by interacting with intracellular DNA sensing pathway. Cell death & disease. 2021;12(4):322.
Koeffler HP, Golde DW. Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity. Science. 1978;200(4346):1153-4.
Breitman TR, Selonick SE, Collins SJ. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proceedings of the National Academy of Sciences of the United States of America. 1980;77(5):2936-40.
Todisco E, Suzuki T, Srivannaboon K, Coustan-Smith E, Raimondi SC, Behm FG, et al. CD38 ligation inhibits normal and leukemic myelopoiesis. Blood. 2000;95(2):535-42.
Casini T, Pelicci PG. A function of p21 during promyelocytic leukemia cell differentiation independent of CDK inhibition and cell cycle arrest. Oncogene. 1999;18(21):3235-43.
Lu Z, Hunter T. Ubiquitylation and proteasomal degradation of the p21(Cip1), p27(Kip1) and p57(Kip2) CDK inhibitors. Cell cycle. 2010;9(12):2342-52.
Liao Y, Liu N, Xia X, Guo Z, Li Y, Jiang L, et al. USP10 modulates the SKP2/Bcr-Abl axis via stabilizing SKP2 in chronic myeloid leukemia. Cell discovery. 2019;5:24.
Agard NJ, Maltby D, Wells JA. Inflammatory stimuli regulate caspase substrate profiles. Molecular & cellular proteomics : MCP. 2010;9(5):880-93.
Sexauer A, Perl A, Yang X, Borowitz M, Gocke C, Rajkhowa T, et al. Terminal myeloid differentiation in vivo is induced by FLT3 inhibition in FLT3/ITD AML. Blood. 2012;120(20):4205-14.
Traer E, Martinez J, Javidi-Sharifi N, Agarwal A, Dunlap J, English I, et al. FGF2 from Marrow Microenvironment Promotes Resistance to FLT3 Inhibitors in Acute Myeloid Leukemia. Cancer research. 2016;76(22):6471-82.
Sakamoto K, Imamura T, Yano M, Yoshida H, Fujiki A, Hirashima Y, et al. Sensitivity of MLL-rearranged AML cells to all-trans retinoic acid is associated with the level of H3K4me2 in the RARalpha promoter region. Blood cancer journal. 2014;4:e205.
Steinman RA. Cell cycle regulators and hematopoiesis. Oncogene. 2002;21(21):3403-13.
Freemerman AJ, Vrana JA, Tombes RM, Jiang H, Chellappan SP, Fisher PB, et al. Effects of antisense p21 (WAF1/CIP1/MDA6) expression on the induction of differentiation and drug-mediated apoptosis in human myeloid leukemia cells (HL-60). Leukemia. 1997;11(4):504-13.
Goswami S, Mani R, Nunes J, Chiang CL, Zapolnik K, Hu E, et al. PP2A is a therapeutically targetable driver of cell fate decisions via a c-Myc/p21 axis in human and murine acute myeloid leukemia. Blood. 2022;139(9):1340-58.
Harris TE, Albrecht JH, Nakanishi M, Darlington GJ. CCAAT/enhancer-binding protein-alpha cooperates with p21 to inhibit cyclin-dependent kinase-2 activity and induces growth arrest independent of DNA binding. The Journal of biological chemistry. 2001;276(31):29200-9.
Coqueret O, Gascan H. Functional interaction of STAT3 transcription factor with the cell cycle inhibitor p21WAF1/CIP1/SDI1. The Journal of biological chemistry. 2000;275(25):18794-800.
Chadha A, Moreau F, Wang S, Dufour A, Chadee K. Entamoeba histolytica activation of caspase-1 degrades cullin that attenuates NF-kappaB dependent signaling from macrophages. PLoS pathogens. 2021;17(9):e1009936.
Gartel AL, Tyner AL. Transcriptional regulation of the p21((WAF1/CIP1)) gene. Experimental cell research. 1999;246(2):280-9.
Ward PS, Patel J, Wise DR, Abdel-Wahab O, Bennett BD, Coller HA, et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer cell. 2010;17(3):225-34.
Figueroa ME, Abdel-Wahab O, Lu C, Ward PS, Patel J, Shih A, et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer cell. 2010;18(6):553-67.
Radomska HS, Basseres DS, Zheng R, Zhang P, Dayaram T, Yamamoto Y, et al. Block of C/EBP alpha function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations. The Journal of experimental medicine. 2006;203(2):371-81.
Mu CF, Shen J, Liang J, Zheng HS, Xiong Y, Wei YH, et al. Targeted drug delivery for tumor therapy inside the bone marrow. Biomaterials. 2018;155:191-202.

No competing interests reported.

SupplementaryData.pdf

Download PDF

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

Cytosolic DNA sensing combined with differentiation therapy induces irreversible differentiation and cell growth arrest in myeloid leukemia cells

Status:

Version 1

Abstract

Figures

Background

Methods

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1