Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

Orlikova-Boyer, Barbora; Lorant, Anne; Gajulapalli, Sruthi Reddy; Cerella, Claudia; Schnekenburger, Michael; Lee, **-Young; Paik, Ji Yeon; Lee, Ye**; Siegel, David; Ross, David; Han, Byung Woo; Nguyen, Thi Kim Yen; Christov, Christo; Kang, Hyoung **; Dicato, Mario; Diederich, Marc

doi:10.1186/s40364-024-00594-w

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

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Published: 04 May 2024

Volume 12, article number 47, (2024)
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Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

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Barbora Orlikova-Boyer¹,
Anne Lorant¹,
Sruthi Reddy Gajulapalli²,
Claudia Cerella¹,
Michael Schnekenburger¹,
**-Young Lee^2,6,
Ji Yeon Paik²,
Ye** Lee²,
David Siegel³,
David Ross³,
Byung Woo Han²,
Thi Kim Yen Nguyen²,
Christo Christov⁴,
Hyoung ** Kang⁵,
Mario Dicato¹ &
…
Marc Diederich²

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Abstract

Background

Despite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.

Methods

Transcriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD⁺ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.

Results

Transcriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.

Conclusions

Overall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

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Introduction

Tyrosine kinase inhibitors (TKIs) represent the frontline treatment against chronic myeloid leukemia (CML), characterized by the t(9:22) chromosomal translocation, resulting in the breakpoint cluster region-Abelson (BCR-ABL)1 fusion oncogene with constitutively activated Abl tyrosine kinase activity. Despite TKI efficacy, resistant mutants turn CML into a controllable yet persisting chronic disease, interfering with the patient’s quality of life [1,2,3]. Moreover, one-third of newly diagnosed patients develop primary or secondary resistance toward approved TKIs, and clinical relapse occurs [4]. Imatinib resistance originates from BCR-ABL-dependent and -independent mechanisms, including leukemia stem cell (LSC) treatment insensitivity. LSCs and TKI/therapy-resistant cells depend on mitochondrial metabolism and oxidative phosphorylation (OXPHOS) to meet their energy demands [5, 6], in contrast to mature CML cells or normal hematopoietic stem cells (HSCs).

The goal of therapeutic approaches for CML treatment is to eliminate LSCs by identifying and targeting genetic alterations and molecular pathways that contribute to their survival and resistance to apoptosis. Efforts must be undertaken to develop combination therapies targeting tyrosine kinases with novel drug candidates that specifically target genes or molecules necessary for the survival mechanisms of CML LSCs [7]. Accordingly, identifying and targeting rewired energy metabolism is a relevant strategy for develo** non-ABL-related therapeutic options for treating TKI-resistant CML patients, preventing the appearance of TKI-resistant cells, and LSC persistence.

Nicotinamide adenine dinucleotide (NAD) is a substrate for NAD⁺-dependent enzymes and a major coenzyme in bioenergetic processes. The NAD⁺/NADH ratio modulates metabolic processes, including oxidative phosphorylation (OXPHOS), tricarboxylic acid (TCA) cycle, fatty acid oxidation (FAO), and glycolysis. NADH serves as a central hydride donor to mitochondrial ATP synthesis. Maintaining NAD⁺ levels and the NAD⁺/NADH ratio is crucial for mitochondrial function, ATP production, and homeostasis. Hence, NAD metabolism is a master regulator linking metabolic processes to OXPHOS [8]. Most cells replenish their NAD⁺ pool primarily via the NAD⁺ salvage pathway by recycling nicotinamide (NAM), generated by NAD⁺-consuming enzymes predominantly by poly (ADP-ribose) polymerase 1 (PARP1) and sirtuin (SIRT)1 [9, 18].

In addition to the cytotoxic activity, whether by apoptosis or controlled necrosis, the activation of the immune-mediated destruction of tumor cells is deeply involved in the curative effect of a given anticancer agent. There is a growing interest in promoting anti-tumor immune responses as a potential alternative strategy for cancer treatment [19]. Accordingly, the induction of immunogenic cell death (ICD) is an attractive approach to fighting cancer malignancies [20]. Pharmacological mediators of ICD stimulate the host’s immune system via the release of damage‐associated molecular patterns (DAMPs), eventually triggering a persistent immune response [21].

We investigated a TKI-independent, immunogenic cell death mechanism triggered by a novel, methylated indolequinone (MAC681). This compound targets PARP1 degradation, exacerbating mitochondrial dysfunction and inducting a non-canonical, necroptotic-like cell death. We used MAC681-treated, dying, or dead cells to demonstrate immunogenic vaccination against myeloid leukemia.

Materials and methods

Computational analysis of public CML datasets

This study utilized several publicly available datasets, which were processed as follows:

A)
GSE5550 [22]: 17 patients’ bone marrow samples sorted for CD34+ hematopoietic stem and progenitor cells, 8 healthy donors, and 9 untreated CML patients in chronic phase were downloaded from GEO. The samples were processed and normalized using the robust Multi-Array Average (RMA) expression measure from the “affy” package in R [23]. All the probes were analyzed separately; if multiple probes representing the same gene were present, only the most significant probe was kept.
B)
GSE97562 [24] and GSE47927 [25]: A total of 19 patients’ bone marrow samples sorted for CD34+, 8 healthy donors, and 11 untreated CML patients in chronic phase raw CEL files were downloaded from GEO (Supp. Table S1A). Samples were processed and normalized using the RMA expression measure from the “affy” package in R [23]. To correct for the batch effect between datasets, we employed the combat function of the "sva" R package [26].
C)
The Microarray Innovations in LEukemia (MILE) study, GSE13159 [27]: A total of 66 untreated CML patients’ bone marrow mononuclear cells raw CEL files were downloaded from GEO. Samples were processed and normalized using the robust RMA expression measure from the “affy” package in R [23]. Samples were divided into 4 groups based on the quartiles of PARP1 expression. The high and low quartiles were subsequently used for the differential expression analysis.
D)
The Cancer Cell Line Encyclopedia (CCLE) dataset: Gene expression transcript per million (TPM) normalized reads from all the cancer cell lines (n = 1165) were downloaded from the Depmap portal website (https://depmap.org/portal/download/all/) [28].

Differential expression analysis

To identify differentially expressed genes (DEGs) between healthy and CML CD34⁺ cells or between CML patients with high and low expression of PARP1, we utilized the “limma” R package [29]. We applied a threshold of |log2(FC)|> 0.3 and a false discovery rate (FDR) < 0.05. The resulting DEGs were visualized using volcano and generated using the “ggplot2” R package [30] (https://ggplot2.tidyverse.org).

Pathways and enrichment analysis

We performed functional enrichment analysis using GO terms, GSE analysis (GSEA), and Reactome pathways to analyze the identified DEGs comprehensively. GO terms and GSEA analysis were performed using “fgsea” and “msigdb” R packages. Reactome enrichments were performed on the web-based application [31] (https://reactome.org/). Pathways were considered significant when the FDR ≤ 0.05. We re-grouped each pathway for its higher hierarchical annotation in Reactome and calculated a weighted percentage of pathways enriched. Genes belonging to the NAD⁺ metabolome pathway were collected from **ing on the nucleus (24 h), a hallmark of necroptosis, are seen (top panel). Mitochondrial alterations (lower panel) induced by MAC681 are also visualized. G Time-dependent induction of mitochondrial membrane potential loss (left panel) and cell death induction (right panel) by MAC681 (5 µM). MMP was analyzed after incubation with MitoTracker Red (100 nM) by flow cytometry. Mitochondrial oxidative phosphorylation uncoupler CCCP (50 µM, 20 min) was used as a positive control. Flow cytometry analyzed cell death induction by propidium iodide (PI) uptake after 15 min staining with PI (0.5 µg/mL). DMSO (50%, 1 min) was used as a positive control (one-way ANOVA, Dunnett's multiple comparisons test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). H Cells were treated with MAC681 (5 µM, 6 h) and analyzed by fluorescence microscopy after staining with Rhod2-AM (Rhod, 2.5 µM), MitoTracker Green (MTG, 100 nM), and Hoechst. The intensity of the Rhod2-AM signal was quantified in regions stained with MitoTracker Green, and the co-occurrence and correlation were quantified by Manders’, Spearman’s, and Pearson’s coefficients. Double positive cells were analyzed, and the average of three independent experiments was shown (double-sided unpaired t-test ***p ≤ 0.001). I Cytosolic Ca²⁺ levels of cells treated with MAC681 (5 µM) for indicated time points, stained by Fluo4-AM, were measured by flow cytometry (double-sided unpaired t-test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). J Cytosolic Ca²⁺ levels of cells treated with MAC681 (5 µM) for indicated time points in the presence of EGTA (650 µM), followed by TSG (10 nM) treatment, were measured by Fluo4-AM every 15 s by flow cytometry, (AUC, double-sided unpaired t-test **p ≤ 0.01, ***p ≤ 0.001). K Colony formation assay with K-562 cells treated with MAC681 at the indicated concentrations. Images are representative of three independent experiments (One-way ANOVA, Dunnett's multiple comparisons test **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). L In vivo zebrafish xenografts model of tumor mass formation after injection of fluorescent CellTracker™ CM-DiI Dye-stained K-562 cells pre-treated for the indicated time. M MAC681 decreases the viability of CML patients’ PBMCs in dose- and time-dependent manners. CML primary cells were treated ex-vivo with indicated concentrations of MAC681, and cell viability was evaluated after 4, 12, and 24 h by trypan blue exclusion test. The histogram corresponds to the mean ± SD (n = 2 CML patients). N Analysis of ALDH activity in K-562 cells. Cells were treated with MAC681 (5 µM, 12 h) or Imatinib (0.25 µM, 24 h), and the ALDH inhibitor diethylaminobenzaldehyde (DEAB) was used to distinguish cell subpopulations with low and high ALDH activity. Representative dot plots showing the percentage of ALDH-positive cells and corresponding quantifications (right panel) of three independent experiments are presented (left panel) (one-way ANOVA, Tukey’s multiple comparisons test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

Full size image

Considering the upregulation of PARP1 in CML patients' datasets and the upregulation of metabolic pathways in patients expressing high levels of PARP1, we wondered if such a strong induction of DNA damage could result in the PARP-dependent depletion of total cellular NAD⁺ levels. Indeed, after MAC681 treatment, we detected an early, time-dependent decrease in cellular NAD⁺. MAC681 treatment reduced NAD⁺ by 20% in 30 min, 59% in 1 h, and 87% in 2 h. NAD⁺ depletion was followed by a reduction in NADH levels. An overall time-dependent decrease in the NAD⁺/NADH ratio was detected (Fig. 2B). Moreover, Hoechst/PI nuclear morphology analysis indicated that MAC681 induced necrotic cell death in K-562 cells and that the PARP1 inhibitor, 3ABA rescued cells from MAC681-induced lethality (Fig. 2C).

As metabolic pathways, including OXPHOS, rely on NAD availability, PARP1 activation promotes a rapid decrease in mitochondrial oxygen consumption [40]. We detected a significantly decreased OCR in MAC681-treated cells compared to the control (Suppl. Fig. S2A). Moreover, in K-562 cells, the basal ATP production rate relied more on oxidative phosphorylation than glycolysis (61% versus 39%, respectively) (Sup. Fig. S2B). Within two hours, MAC681 treatment induced a 43% reduction in the total ATP production rate compared to untreated cells (Fig. 2D). Furthermore, the MAC681-induced decrease in the ATP production rate was mainly due to decreased mitochondrial ATP production rate (Fig. 2D). Subsequently, after 4 h of treatment, we observed a 53.2% reduction in total intracellular ATP levels. After 8 h, ATP levels were reduced by 92% compared to controls (Fig. 2E).

The loss of NAD⁺ precedes the induction of mitochondrial depolarization and mitochondrial outer membrane permeability transition [41]. TEM analysis confirmed time-dependent mitochondrial alterations after MAC681 treatment, disrupting mitochondrial cristae after 4 h and mitochondrial swelling after 8 h (Fig. 2F, lower panel). In addition, the cells adopted a necroptotic phenotype (Fig. 2F, upper panel). In agreement with morphological changes and ATP loss, MAC681 triggered a time-dependent loss of mitochondrial membrane potential (MMP) (Fig. 2G, left panel). The MMP was strongly reduced at 4 h and 8 h of treatment, although cells remained viable, considering the minimal PI staining intake (Fig. 2G, right panel).

Since mitochondrial calcium overload could have triggered these mitochondrial morphological alterations, we assessed calcium accumulation in mitochondria by co-localizing Rhod2-AM mitochondrial Ca²⁺ dye with MitoTracker green after 6 h of treatment. Our results indicate that MAC681 induced Ca²⁺ accumulation in 67% of the mitochondria (Fig. 2H and Sup. Fig. S2C). To further analyze the perturbation of calcium homeostasis, we assessed the effect of MAC681 on cytosolic Ca²⁺ accumulation after Fluo4-AM staining. We detected an immediate increase in cytosolic Ca²⁺ at 2 h, which decreased after 4 h and returned to basal values at 8 h. At 10 h, we observed a secondary increase in cytosolic Ca²⁺ (Fig. 2I).

To further ascertain the origin of the observed cytosolic Ca²⁺ accumulation, we used EGTA to chelate extracellular Ca²⁺ influx. Under these conditions, adding the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (TSG) led to a rapid accumulation of cytosolic Ca²⁺ in control cells. Interestingly, in MAC681-treated cells for 2 h, the addition of TSG significantly reduced the increase, which was abolished at 4 h (Fig. 2J). These data show that MAC681 rapidly depleted endoplasmic reticulum Ca²⁺ in line with the observed initial cytosolic Ca²⁺ increase.

PARP1 activation after DNA damage contributes to mitochondrial Ca²⁺ dysregulation and subsequent calpain activation [42, 43] to promote truncation and translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus. In MAC681-treated cells, AIF co-localized with nuclear Hoechst staining (Sup. Fig. S2D-E) compared to untreated controls. Moreover, we observed that calpain inhibitor PD 150606 prevented the PARP1 degradation (Sup. Fig. S2F), suggesting the involvement of calpain activation during MAC681-induced metabolic catastrophe.

The translational potential of MAC681 as an antileukemic agent in vitro and in vivo

Considering the observed antimetabolic potential of the methylated indolequinone MAC681, we assessed its effects on the viability in four human leukemia cells (K-562, K-562R, Jurkat, and U-937) (Sup. Fig. S2G). To assess differential toxicity, we treated the non-cancerous, proliferating cell line RPMI1788 representing healthy B lymphocytes and PBMCs obtained from healthy donors with increasing concentrations of MAC681. RPMI 1788 cells were 65-fold and PBMCs 51-fold less affected compared to K-562 cells when treated at 10 µM after 72 h (Sup. Fig. S2H).

Considering the above-mentioned antileukemic effect of MAC681 at low µM concentrations, we investigated the effect of MAC681 by CFA. Results showed a dose-dependent decrease in the colony formation capacity of three chronic myeloid leukemia cell lines (K-562, KBM-5, Meg-01) and two acute myeloid leukemia cell lines (U-937, HL-60) (Fig. 2K and Sup. Fig. S2I-J). Next, we extended CFA results toward a zebrafish xenograft model to provide a more solid basis for the translational value of MAC681. In vivo data showed time-dependent inhibition of tumor mass formation (Fig. 2L and Sup. Fig. S2K). Moreover, we treated ex-vivo primary CML cells from two CML patients with MAC681. As seen in Fig. 2M, the MAC681 efficiently reduced the viability of CML patients’ PBMCs in dose- and time-dependent manners. We next investigated if MAC681 could target ALDH-positive cells. High expression of ALDH has been reported for normal and cancer stem and progenitor cells of various lineages, including hematopoietic cells [44, 45]. Elevated ALDH activity is an established marker for the identification of hematopoietic cells with stem-like characteristics. Our data shown in Fig. 2N provide evidence that MAC681 (5 µM, 12 h) is targeting preferentially the ALDH-positive population, decreasing the amount of ALDH bright cells by 93.3%. Interestingly, imatinib treatment (0.25 µM) at 24 h reduced the ALDH-positive population only by 42.4%. Our data validated MAC681 as an attractive antileukemic agent for further mechanistic investigations.

Off-target prevention of MAC681-induced cell death by the PARP inhibitor 3-aminobenzamide

As we previously detected a protective effect of the PARP inhibitor 3ABA against MAC681-induced cell death (Fig. 2C), we next assessed the effect of the 3ABA pre-treatment on the PARP1 protein level. Surprisingly, we observed the disappearance of full-length PARP1 (116 kDa) without any cleaved fragments after 8 h of MAC681 treatment alone. 3ABA pre-treatment prevented this PARP1 reduction (Fig. 3A). Hence, we analyzed the kinetics of the atypical MAC681-induced PARP1 reduction (Fig. 3B). We used an additional antibody able to recognize both apoptotic as well as necrotic PARP1 cleaved fragments (Supp. Fig. S3A). However, we did not detect any cleavage fragments either. In addition, we observed that MAC681 induced a time-dependent downregulation of PARP1 mRNA expression levels, which is prevented in the presence of 3ABA (Supp. Fig. S3B). Next, we verified whether the disappearance of full-length PARP1 might be related to its low mRNA levels after MAC681 treatment. Accordingly, we analyzed the stability of PARP1 in the presence of cycloheximide that blocks the elongation phase of protein synthesis (Supp. Fig. S3C). We showed that the stability of PARP1 protein remains unchanged up to 24 h in the presence of cycloheximide, proving that the PARP1 protein degradation observed after MAC681 treatment is independent of its effects on PARP1 mRNA levels.

This type of atypical degradation could be explained by the direct interaction of MAC681 with PARP1 protein, a hypothesis we validated by computational docking. The control docking showed that 2, 3-dihydrobenzofuran-7-carboximade derivative (2US) was docked into the catalytic region of PARP1 in good agreement location with the actual binding mode in the crystal structure of PARP1 in complex with 2US (PDB ID: 40QA) (Supp. Fig. S3D, upper panel). We demonstrated that MAC681 binds with high affinity into the catalytic region of the PARP1 structure in the presence of damaged DNA (Supp. Fig. S3D, lower panel, MAC681 in magenta). MAC681 was located in active sites of the PARP1 with stabilization energies of − 7.1 kcal/mol. We closely observed that the aromatic ring of the bound MAC681 is surrounded by well-known inhibitor-binding residues such as His862, Tyr889, Tyr896, Phe897, and Tyr907 (Fig. 3C).

We further focused on the protective role of 3ABA. We assessed its effects on MAC681-induced ATP depletion and mitochondrial membrane potential (MMP) loss leading to cell death as analyzed by PI intake. 3ABA alone did not affect cellular ATP levels. On the other hand, 3ABA pre-treatment restored ATP levels that were depleted after the MAC681 treatment (Fig. 3D). Similarly, 3ABA alone did not affect MMP. It partially reverted the loss of MMP compared to cells treated with MAC681 alone (Fig. 3E left panel). Moreover, 3ABA rescued the cells from dying, as shown by PI intake (Fig. 3E right panel).

3ABA prevented the DNA damage induced by MAC681 not only in K-562 cells (Fig. 3F) but also in U-937, MOLM-13, MV4-11, and OCI-AML3 cell lines (Supp. Fig. S3E left panel) where MAC681 induced typical apoptotic nuclear fragmentation patterns (Supp. Fig. S3E right panel). 3ABA prevented observed nuclear morphology changes in all cell models. Concomitantly, 3ABA counteracted the characteristic apoptotic fragmentation of PARP1 protein caused by MAC681 treatment in U-937 cells (Supp. Fig. S3F). Interestingly, this protection (of cell death induction and PARP1 fragmentation) by 3ABA could not be observed in apoptotic U-937 cells treated with etoposide (Supp. Fig. S3F), demonstrating the specificity of a combination involving MAC681 and 3ABA. These data indicate that the protective effect of 3ABA in MAC681 treated cells could be independent of its primary PARP1-inhibition function and instead mediated by its so far not-described off-target effect.

The chemical structures of MAC681 and 3ABA are recognized as suitable reaction partners. Based on our previous results, the chemical structure of 3ABA seemed to be directly involved in the protective effect. Thus, we hypothesized that indolequinone MAC681 could act as a Michael acceptor, bearing the α,β-unsaturated bond, reacting with the aniline nucleophile of 3ABA (Michael donor), able to attack the position next to the -OCH₃ of the quinone ring of MAC681. The above-mentioned Michael reaction would result in a hypothetical 3ABA-MAC681-5-position adduct (Fig. 3G). On the other hand, the reaction between 3ABA and MAC681 can also give rise to the 6-position adduct due to the loss of the methoxy group with the addition of the amine and the 2,4,6-trifluorophenol part of the molecule either intact (Fig. 3G) or lost (not shown). Using mass spectrometry, we confirmed the presence of a 6-position adduct when cells were treated with a combination of MAC681 and 3ABA for 6 h (Supp. Fig. S3G). Moreover, the 3ABA washout completely abrogates the protective effect of 3ABA detected without washout (Suppl. Fig. S3H). In the washout group, cells pre-treated with 3ABA and treated with MAC681 for 4 h were losing ATP at the same extent as cells treated with MAC681 alone (S3H upper left panel). The loss of the protection by 3ABA on early effects mediated by MAC681 was confirmed by the late outcome on cell concentration (S3H upper middle panel) and viability (S3H upper right panel). In addition, we confirmed that the cells in the washout group (3ABA + MAC681) were dying by the same cell death modality as cells treated with MAC681 alone, as shown by identical nuclear morphology patterns (S3H lower panel).

Indolequinones' DNA damaging potential is linked to their redox-cycling activity or alkylation potential. Using dichlorofluorescein and redox sensor red probes, we confirmed that MAC681 did not induce any reactive oxygen species (ROS) (Supp. Fig. S3I) and thus did not act as a redox cycler. Considering its alkylation activity, MAC681 could be transformed into a DNA alkylation agent through bioreductive activation by two electron reductases, such as NAD(P)H quinone dehydrogenase (NQO)1 or NQO2, via mechanisms of iminium salt (Supp. Fig. S3J) [46] or quinone methide formation (Supp. Fig. S3K) [47]. Analysis of CML patient datasets showed that CML patients have significantly higher expression of NQO1 or NQO2 or both than their healthy counterparts (Supp. Fig. S3L). Moreover, based on the CCLE dataset, K-562 cells have the highest expression of NQO2 and the second highest expression of NQO1 within the CML cell lines available (Supp. Fig. S3M).

Considering this, the above-mentioned hybrid compounds (5-position and 6-position adducts) would reduce the overall DNA damaging activity of MAC681 and thus confer 3ABA-mediated protection against MAC681-induced controlled necrosis triggered by excessive DNA damage.

In addition, docking results revealed that 3ABA and both hybrid compounds (3ABA-MAC681-5-position adduct and 3ABA-MAC681-6-position adduct) docked well into the similar PARP1 catalytic region where MAC681 did. Interestingly, both hybrid compounds have bound to PARP1 tighter than MAC681 or 3ABA alone, with stabilization energies of -9.4 and -8.5 kcal/mol, respectively (Fig. 3H).

MAC681-induced cell death has immunogenic potential

Our initial analysis of transcriptomic data (GSE5550) showed a significant down-regulation of genes encoding proteins involved in adaptive and innate immune responses in CML patients (Fig. 4A). Especially genes leading the NES score, belonging to antigen processing and presentation and interferon-gamma signaling categories are down-regulated (Fig. 4A). As the attenuation of these essential immune recognition mechanisms reduces the capacity of the patient’s immune effectors to recognize and eliminate the accumulating CML blasts, we were interesting to characterize further the MAC681-induced necrotic cell death observed in K-562 cells from the immunogenic perspective. We measured the emission of ICD-related DAMPs, including calreticulin exposure, extracellular ATP secretion, and HMGB1 release. As soon as 10 min and up to 2 h after the treatment, MAC681-treated cells reached between 2- to fourfold increase in ATP secretion compared to untreated controls (Fig. 4B). Additionally, we determined that 10 h of 5 µM MAC681 treatment doubled the exposure of CRT at the cell surface compared to the control (Fig. 4C). Besides intrinsic cellular mechanisms, the translocation of calreticulin is also mediated by soluble factors that operate in an autocrine/paracrine manner. Accordingly, IL-8 can be hyperactivated by ICD-inducers to facilitate the immunogenic exposure of calreticulin [48]. Hence, we measured the human IL-8 concentrations in cell culture supernatants and showed that 5 µM of MAC681 significantly induced IL-8 release at 8 and 10 h of treatment compared to control (1.4- and 1.3-fold increase, respectively) (Fig. 4D). Timeframe of IL-8 release is in line with calreticulin cell surface exposure in K-562 cells. In addition, we also provided evidence showing that at 8 h, MAC681 induced IL-8 mRNA expression in a dose-dependent manner in K-562 cells with a 6.4-fold increase at 5 µM (Supp. Fig. S4A).

Furthermore, we showed that necrosis-inducing concentration triggered significant HMGB1 release starting from 4 h and peaking at 24 h in MAC681-treated K-562 cells (Fig. 4E). Moreover, we showed that MAC681-induced HMGB1 release not only in CML cells but also in OCI-AML3 and C1498 AML cell lines (Supp. Fig. S4B-C). As the emission of DAMPs by dying cells plays a crucial role in recruiting immune effectors, we co-cultured J774A1 macrophages with MAC681 pre-treated K-562 cells. Fluorescence microscopy showed a 160-fold increase in the engulfment of red-stained MAC681-treated K-562 cells by green-stained macrophages (Fig. 4F).

However, neither morphological assessments of dying cells nor the release of the above-mentioned biochemical correlates, alone or in combination, can predict with complete certainty the induction of bona fide ICD [49]. The gold-standard approach to proving the immunogenicity of the cellular demise induced by a specific agent relies on vaccination experiments in immunocompetent mice. Hence, we treated syngeneic murine myeloid C1498 cancer cells with the MAC681 at different concentrations for 24 h and evaluated cell death induction by flow cytometry after annexin V/PI staining (Supp. Fig. S4D). Next, syngeneic immunocompetent C57BL/6 mice were vaccinated with dying cancer cells treated either with 5 µM MAC681 or a combination of 2.5 µM cytarabine (CTB) and 0.5 µM doxorubicin (DOX), a known induction therapy, into the right flanks. Control mice (non-vaccinated) were given 1 × PBS only. After one week, all mice were challenged with living C1498 cells into the contralateral flank of the mice, and tumor growth was monitored until the tumor volume reached > 600 mm³ in the non-vaccinated control group. We showed that tumor volume and weight were significantly reduced in the MAC681-treated group compared to the control group (Fig. 4G). Moreover, the MAC681 effect on tumor growth was similar to the impact of the induction therapy (CTB + DOX). In addition, we assessed the effect of MAC681 vaccination on spleen morphology and revealed that MAC681 significantly reduced spleen weight but did not impact its length (Fig. 4H). Altogether, these data provide strong evidence supporting the immunogenic potential of indolequinone MAC681.

Synergistic potential of MAC681 in combination with asciminib in imatinib-sensitive and -resistant human leukemia

Despite improvements in CML therapy by develo** efficient TKIs suppressing BCR-ABL activity, one in five patients develops resistance to TKIs. Moreover, 22% of de novo CML- patients in the chronic phase become intolerant (JALSG CML212 study), requiring discontinuation of the therapy because of adverse side effects. Accordingly, combining TKIs with non-ABL-related therapeutic strategies is required to prevent the onset and overcome the appearance of TKI-resistant clones or diminish the side effects of TKIs. Hence, we investigated the effect of the combination of MAC681 with the ATP-non-competitive, allosteric TKI asciminib in multi-TKI-resistant and sensitive K-562 cell models up to 72 h. We used the Chou-Talalay algorithm to determine the combination index and fraction-affected values. Our results in K-562R cells showed a synergistic effect of 3 µM MAC681 with 1, 10, and 30 µM of asciminib at any given time point (Fig. 5A). In K-562 sensitive cells, multiple combinations showed synergistic effects during 24 h, 48 h, and 72 h (Fig. 5B).

Discussion

CML cells express the BCR-ABL1 fusion oncogene, which leads to profound deregulation of the gene expression and, consequently, protein expression of leukemic cells. These deregulations lead to ROS production, DNA repair pathway deregulation, and a metabolic adaptation conferring proliferation and survival advantage. CML cells are particularly addicted to these forms of metabolic and functional dysregulations. CML cells are in high energy demand, and metabolic perturbations lead to insufficient energy production. Pharmacologic intervention can address this metabolic state characterized by increased glycolytic metabolism. Here, we demonstrate the capacity of the indolequinone MAC681 to induce metabolic catastrophe followed by the induction of non-canonical controlled necrosis to “kill unkillable cells” [50].

Targeting these above-mentioned essential pathways in addition to TKI-mediated interference with BCR-ABL1-driven signaling pathways is considered a promising antileukemic approach. We previously targeted the accumulation of ROS in CML cells with the hydroquinone TMQ0153, leading to the accumulation of intracellular ROS levels followed by ATP loss, autophagy, leading concentration-dependently to apoptosis, and controlled necrosis [51]. Whereas normal HSCs and mature CML cells rely predominantly on glycolysis [6], CML LSCs use oxidative phosphorylation for survival [5]. However, there is conflicting information regarding the energy metabolism of TKI-resistant CML cells. Patel et al. showed that stem cells from non-mutational resistant patients rely on glycolysis due to a progressive failure of the electron transport chain. This metabolic adaptation was attributed to STAT-3-mediated reprogramming of the mitochondrial metabolism [52]. Accordingly, the use of metabolic inhibitors, including the inhibitor of glucose metabolism by 2-Deoxy-d-glucose (2-DG), reduced CML cell viability, and a co-treatment of 2-DG with imatinib led to synergistic autophagic cell death [53]. Interestingly, in response to imatinib, BCR-ABL-driven human leukemia cells significantly reduced glucose consumption and lactate secretion and increased intracellular ATP levels, indicating a switch from glycolysis to mitochondrial oxidative phosphorylation [54, 55].

Hence, unsurprisingly, other groups provided evidence that treatment-resistant CML cells appear to be OXPHOS-dependent [5, 8]. Similar to the latter, comparing the metabolic profile between K-562 sensitive and K-562R cells in our lab, we detected that multi-resistant K-562 cells display a higher OXPHOS metabolism than the parental K-562 (data not shown).

Moreover, patient cells resistant to imatinib presented differential levels of amino acids and acylcarnitines [56], accumulation of citric acid intermediates, and increased levels of NADH [57], altogether witnessing a profound rewiring of the mitochondrial metabolic capacity and underlining the essential role of mitochondrial metabolism in CML resistance. As leukemic cells are addicted to NAD for proliferation and survival, we postulate that new drug entities targeting mitochondrial physiology and reducing NADH levels can act as potent cytotoxic agents against sensitive and resistant CML blasts. Consequently, MAC681 reduced the NADH levels, contributing to reduced ATP production and subsequently controlled necrosis (Fig. 2B-F).

DNA damage repair is a biological process deregulated in CML, providing advantages and disadvantages for CML blasts [58]. Indeed, the rapid proliferation rate of CML cells requires rapid cell cycle progress without extensive pausing in S and early G2 phases due to DNA repair. Accordingly, using rapid alternative nonhomologous end-joining (ALT-NHEJ), CML cells achieve a growth advantage despite the accumulation of mutations, eventually leading to genomic instability and chromosomal condensation [24]. Targeting additional components of the faulty CML DNA repair pathways, for example, by inhibiting the essential DNA repair factor PARP1, could constitute a helpful therapeutic strategy to be exploited by single or combined treatments, especially as PARP1 and DNA ligase IIIα levels were described as biomarkers in resistant CML patients. Targeting the ALT-NHEJ pathway when treatment with TKIs failed was previously targeted by L67, a DNA ligase I and IIIα inhibitor, and the PARP1 inhibitor NU1025 [13]. In opposition to our data showing a complete degradation of PARP1 by MAC681 alone, none of these agents had the potential to affect CML cells. The authors showed, nevertheless, a specific sensitivity of resistant CML cell lines when combined, validating the strategy of targeting the ALT-NHEJ pathway as an interesting therapeutic target, not only in CML but also in other cancers relying on the same mechanisms. Considering that imatinib treatment could reprogram DNA repair in the first days of therapy [59], we postulate that a combined treatment by ALT-NHEJ inhibitors and BCR-ABL1 inhibitors could be particularly efficient and prevent the emergence of resistance at later time points. In that sense, we assessed the effect of combined treatment by the PARP1-inhibiting indolequinone MAC681 and asciminib, belonging to the specifically targeting the ABL myristoyl pocket anti-BCR-ABL1 drug category. So far, few combination treatments against CML involving asciminib have been published. Synergistic interactions of asciminib with imatinib or dasatinib had been observed in KCL-22 CML cells, whereas other authors observed antagonism between asciminib and the other drugs [60]. Interestingly, from an immunologic point of view, T cells treated with asciminib and other TKIs maintained their metabolic plasticity, witnessing metabolic fitness and flexibility. Moreover, asciminib presented a more moderate inhibitory effect of T cell activation, beneficial for controlling CML by immune effectors [61]. Here, we show the first synergetic induction of cell death by MAC681 combined with asciminib, opening new windows for innovative anti-CML treatments.

Resistance to physiological and tolerogenic apoptosis is widely considered a cancer and CML resistance factor. Therefore, searching for pharmacologically active compounds leading to non-canonical forms of cell death is essential. MAC681 belongs to this category of pre-clinically assessed drug candidates. We observed rapid loss of ATP and NADH, accompanied by DNA damage accumulation and progressive degradation of the mitochondrial integrity, leading to cell death that was sensitive to 3-aminobenzamide. The late release of truncated AIF is reminiscent of parthanatos, a form of PARP-1-dependent cell death involving the generation of PAR followed by the nuclear translocation of AIF, resulting in caspase-independent cell death [62]. We were unable to show hyperparylation (data not shown). In contrast, we consider that the late release of AIF from the mitochondria is a consequence of mitochondrial degradation rather than a regulated step of parthanatos.

Targeting the metabolism of myeloid leukemia is a promising therapeutic approach. The small molecule CCI-006 was described as a novel inhibitor of mixed lineage leukemia (MLL)-rearranged, and CALM-AF10 translocated myeloid leukemia cells by targeting a metabolic vulnerability in a subset of low HIF1alpha/low MEIS1-expressing MLL-rearranged leukemia cells that present more glycolytic metabolic phenotype [63].

NQO1 appears to be an essential and central regulator of cellular metabolism, as a perturbation of this enzymatic function by indolequinones may contribute to mitochondrial dysfunction. In opposition to MAC681, the compound KL1333 reacts with NQO1 as a substrate, increasing intracellular NAD⁺ and ATP levels. As a result, this compound decreased lactate and ROS levels and increased mitochondrial mass, membrane potential, and oxidative capacity [64], demonstrating the profound metabolic effect of positive and negative modulation of NQO1. β-Lapachone is bioactivated by NQO1 by a mechanism different from MAC681, as β-lapachone triggers a futile redox cycle that consumes oxygen and generates high levels of ROS. As a result, β-lapachone treatment leads to DNA damage and rapid PARP1-mediated NAD⁺ consumption. These results show that NAD depletion is an essential mechanism to kill leukemia cells with particularly elevated metabolic needs. A combined NAD(P)⁺ depletion and inhibition of glycolysis reduces ATP levels similar to MAC681 treatment. Moore et al. further exacerbated this effect by using the nicotinamide phosphoribosyl transferase (NAMPT) inhibitor FK866, which contributed to an even stronger depletion of NAD⁺ pools as a strategy to target NQO1⁺ cancer types [65]. Chen et coll. described NQO1-mediated necrosis as noptosis in the case of β-lapachone-induced cell death [66]. Considering the essential contribution of ROS to noptosis initiation, we believe that MAC681-induced controlled necrosis differs in part from noptosis, considering the absence of redox cycling. In agreement with Chen et coll., we believe NQO1 remains an essential regulator of non-canonical cell death modalities.

3ABA is the most used first-generation PARP inhibitor. It is known to enhance radiation-induced cell death by sensitizing cancer cells to radiation and, as such, augmenting the antitumor efficacy [67, 68]. Moreover, using 3ABA in combination with DNA-damaging drugs became an attractive strategy against resistant cancer cells to enhance the apoptotic activity of these drugs [69,70,71,72]. Despite its non-toxic properties and initial high specificity towards PARP, some off-target effects have been reported in mammalian and plant systems. Several studies demonstrated its PARP-independent mechanisms, including inhibition of microbe-associated molecular patterns (MAMPs)-induced callose deposition in plants [73], indirect inhibition of protein kinase C in U-937 cells [74] or its antioxidative potential as a hydroxyl radical scavenger in the cell-free system of the rat brain cortex [75]. In addition, 3ABA mitigated chemically-induced toxicity by inhibiting a cytochrome P450-dependent metabolic activation of dichlobenil in the mouse olfactory mucosa [76]. However, we present evidence of a novel off-target effect of 3ABA for the first time. Due to its ability to act as a reaction partner (such as a Michael donor), 3ABA could paradoxically function as an antagonist and decrease the efficacy of the anticancer drugs when used in combinatory treatment. In general, many α,β-unsaturated compounds possess cytotoxic properties as DNA might get alkylated by attacking the β carbon of such compounds. Hence, once 3ABA interacts with indolequinone MAC681, it loses its ability to cause detrimental DNA damage. This discovery is important considering that 3ABA is abundantly used as a bona fide PARP1 inhibitor, and prior evaluation of the chemical structures of the compounds selected for the combinatory studies with 3ABA is necessary to exclude candidates that would fit Michael acceptor characteristics or other possible reaction-partners.

MAC681 has a dual impact, inducing DNA damage while concurrently blocking its repair. We describe here that the prevention of PARP1 degradation by an inhibitor, which blocks its repair activity, should not block the initiation of DNA damage. Our experimental findings indicate that the protective effect of 3ABA is not solely attributed to preventing the degradation of PARP1 but is likely a consequence of its role in preventing MAC681-induced DNA damage (Fig. 3F).

For this study, we recognize the distinct nature of CML and AML as separate diseases, yet they share a common hematological myeloid differentiation path. The cell lines used represent different stages of this path, and our intention was to demonstrate the broader applicability of MAC681 across the myeloid differentiation pathway. Recent advances in genomics and molecular biology have revealed a complex landscape of genetic and molecular alterations in myeloid leukemia. These findings underscore the heterogeneity of the disease and the fluid nature of its classification as new molecular subtypes continue to be identified. Despite potential mechanistic differences, the MAC681's ability to induce immunogenic cell death and exhibit anti-leukemic effects in both leukemia types highlights its promising therapeutic potential across various myeloid malignancies.

Altogether, we present a pharmacological approach leading to the deletion of PARP1, an essential cofactor for DNA repair. We exploit leukemia-intrinsic DNA damage response defects known to induce immunogenicity phenotypes likely to lead to activation of the immune cycle. Indeed, PARP1 inhibition could lead to immunogenic neoantigen accumulation, presented on the surface of dying CML cells, recognized by the immune system to improve immune recognition of cells otherwise poorly immunogenic. Our results show, in addition, downregulation of the immune proteasome as well as likely reduced antigen presentation capacity of the patient’s CML cells. This effect is further amplified by ROS accumulation and mitochondrial integrity disruption as mitochondrial DAMPs, known to play a nefarious role in chronic inflammation, could contribute to improving immune recognition of dying CML cells by immune effectors. The similarity between mitochondria and bacteria was evoked to understand the activation of innate immune mechanisms [77]. We validated the release of DAMPs and the increased immunogenicity of myeloid leukemia cells by an in vivo vaccination approach, achieving a significantly reduced tumor growth after vaccination with dying myeloid cells. We predict that this type of vaccination could be further improved by using immune checkpoint inhibitors. Altogether, increasing the tumor mutational burden by PARP1 inhibition and mitochondrial deregulation could add CML to the cancer types that could benefit from immunotherapy.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

3ABA:: 3-Aminobenzamide
ABL:: Abelson
AIF:: Apoptosis-inducing factor
AML:: Acute myeloid leukemia
ALT-NHEJ:: Alternative nonhomologous DNA end joining
ATCC:: American type culture collection
BCR:: Breakpoint cluster region
BRCA:: Breast cancer gene
CCCP:: Carbonyl cyanide 3-chlorophenylhydrazone
CCLE:: Cancer Cell Line Encyclopedia
CHX:: Cycloheximide
CI:: Combination index
CML:: Chronic myeloid leukemia
cNHEJ:: Classic nonhomologous DNA end joining
CTB:: Cytarabine
DAMPs:: Damage-associated molecular patterns
2-DG:: 2-Deoxy-d-glucose
DEGs:: Differentially expressed genes
DMSO:: Dimethyl sulfoxide
DNA-PK:: DNA-dependent protein kinase
DOX:: Doxorubicin
DSMZ:: Deutsche Sammlung für Mikroorganismen und Zellkulturen
FAO:: Fatty acid oxidation
FBS:: Fetal bovine serum
FC:: Fold change
FCS:: Fetal calf serum
FDR:: False discovery rate
FITC:: Fluorescein isothiocyanate
GO:: Gene ontology
GSE:: Gene set enrichment
GSEA:: GSE analysis
HMGB1:: High mobility group box 1
HR:: Homologous recombination
HSC:: Hematopoietic stem cell
ICD:: Immunogenic cell death
IL:: Interleukin
K-562R:: Imatinib-resistant K-562
LSC:: Leukemia stem cell
Mcl-1:: Myeloid cell leukemia 1
MFI:: Median fluorescent intensity
MLL:: Mixed lineage leukemia
MPER:: Mammalian protein extraction reagent
NAD:: Nicotinamide adenine dinucleotide
NAM:: Nicotinamide
NES:: Normalized enrichment score
NQO:: NAD(P)H quinone dehydrogenase
OCR:: Oxygen consumption rate
OXPHOS:: Oxidative phosphorylation
PARP:: Poly(ADP-ribose) polymerase
PBMC:: Peripheral blood mononuclear cell
PBS:: Phosphate-buffered saline
PDB:: Protein data bank
PMSF:: Phenylmethylsulfonyl fluoride
PI:: Propidium iodide
qPCR:: Quantitative PCR
RT-PCR:: Reverse transcription polymerase chain reaction
ROS:: Reactive oxygen species
SDS:: Sodium dodecyl sulfate
SERCA:: Sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase
siRNA:: Small interfering RNA
SIRT:: Sirtuin
TCA:: Tricarboxylic acid cycle
TKI:: Tyrosine kinase inhibitor
TMB:: Tetramethylbenzidine
TSG:: Thapsigargin
VP-16:: Etoposide

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Acknowledgements

The authors thank Professor Gilbert Kirsch (University of Lorraine, France) for his insights on chemical questions that enhanced our research. DS and DR acknowledge the assistance of Dr. Michael Armstrong in the University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences Mass Spectrometry Facility.

Funding

Research at LBMCC was supported by the “Recherche Cancer et Sang” foundation, the “Recherches Scientifiques Luxembourg” association, the “Een Häerz fir kriibskrank Kanner” association, the Action LIONS “Vaincre le Cancer” association, and Télévie Luxembourg. A grant from Télévie-Luxembourg supported BO-B and AL. CCe and MS were partly supported by the Waxweiler grant of the Action LIONS Vaincre le Cancer. MD was supported by the National Research Foundation (NRF) [Grant Number 2022R1A2C101314111] and by a grant from the MEST of Korea for Tumor Microenvironment Global Core Research Center (GCRC) [Grant Number 2011–0030001]. BWH was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2019R1A2C1090251 and RS-2023–00218543) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HP23C0102 to BWH). Support from Brain Korea (BK21) FOUR program and Creative-Pioneering Researchers Program at Seoul National University [Funding number: 370C-20160062] are also acknowledged.

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Authors and Affiliations

Laboratoire de Biologie Moléculaire du Cancer, BAM3 Pavillon 2, 6A Rue Nicolas-Ernest Barblé, L-1210, Luxembourg, Luxembourg
Barbora Orlikova-Boyer, Anne Lorant, Claudia Cerella, Michael Schnekenburger & Mario Dicato
Department of Pharmacy, Research Institute of Pharmaceutical Sciences & Natural Products Research Institute, College of Pharmacy, Seoul National University, 1, Gwanak-Ro, Gwanak-Gu, Seoul, 08826, Republic of Korea
Sruthi Reddy Gajulapalli, **-Young Lee, Ji Yeon Paik, Ye** Lee, Byung Woo Han, Thi Kim Yen Nguyen & Marc Diederich
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA
David Siegel & David Ross
Faculté de Médecine, Université de Lorraine, Nancy, France
Christo Christov
Department of Pediatrics, Seoul National University College of Medicine, Seoul National University Cancer Research Institute, Seoul National University Children’s Hospital, Seoul, 03080, Republic of Korea
Hyoung ** Kang
Present address: Department of Biological Sciences, Keimyung University, Daegu, 42601, Republic of Korea
**-Young Lee

Authors

Barbora Orlikova-Boyer
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Anne Lorant
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Sruthi Reddy Gajulapalli
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Claudia Cerella
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Michael Schnekenburger
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**-Young Lee
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Ji Yeon Paik
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Ye** Lee
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David Siegel
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David Ross
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Byung Woo Han
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Thi Kim Yen Nguyen
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Christo Christov
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Hyoung ** Kang
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Mario Dicato
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Marc Diederich
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Contributions

BO-B performed, analyzed, and interpreted in vitro data. AL performed and analyzed computational analyses. SRJ and JYL performed animal experiments. CCh supervised and interpreted electron microscopy experiments. HJK and JYP provided and analyzed CML patient samples. DS and DR performed HPLC-MS experiments in collaboration with the University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences Mass Spectrometry Facility. BWH and NTKY conducted electron molecular docking experiments. CCe, MS, and YL assisted with in vitro experimental processes. BO-B, AL, CCh, CCe, MS, MDic, and MDie interpreted data. BO-B, AL, and MDie wrote the manuscript. MDic and MDie supervised and funded the study. All authors read and approved the final manuscript.

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Correspondence to Marc Diederich.

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Ethics approvals and consent to participate

Mice were maintained according to the Institutional Animal Care and Use Committee (IACUC) standards followed by the Seoul National University, South Korea. This study was approved by Seoul National University IACUC (SNU-211222–5).

The zebrafish study was conducted in agreement with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU- 191218–5-1).

Human PBMCs were obtained from Red Cross Luxembourg with informed consent from all subjects (donors of PBMC blood products) involved in the study [LBMCC- 2019–0001].

For human leukemia samples, the Institutional Review Board of Seoul National University Hospital reviewed and approved the study protocol and exempted the study from the obligation to obtain informed consent. This study was performed following the World Medical Association's Declaration of Helsinki.

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The authors declare no competing interests.

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Supplementary Information

Additional file 1: Supplementary Figure 1.

[S1] Voronoi diagram of the superpathway and its children, yellow indicates matched entities and gradient represents p-value for [A] GSE5550, [B] Combined dataset.

Additional file 2: Supplementary Figure 2.

[S2A] MAC681 treatment reduces the basal oxygen consumption rate in K-562 cells (double-sided unpaired t-test **p ≤ 0.01). [S2B] K-562 cells depend more on mitochondrial ATP than glycolytic ATP production. MAC681 treatment specifically reduces the mitochondrial ATP production rate (double-sided unpaired t-test **p ≤ 0.01). [S2C] Mitochondrial Ca²⁺ co-localization was confirmed by the calculation of Manders' coefficients (co-occurrence), Pearson's coefficient (correlation), and Spearman's coefficient (correlation). [S2D] Time-dependent translocation of AIF from mitochondria to the nucleus induced by MAC681 (5 µM). Immunofluorescence microscopy analysis of K-562 cells treated or not with MAC681, incubated with the AIF antibody, and counter-stained with Hoechst. Representative pictures from three independent experiments are shown. [S2E] Time-dependent translocation of AIF from the cytoplasm to the nucleus in K-562 cells, assessed by western blot. Tubulin and lamin B were used as loading controls. After quantifying the bands of interest, cytoplasmic and nuclear proteins were normalized to tubulin and lamin B, respectively (double-sided unpaired t-test *p ≤ 0.05, ****p ≤ 0.0001). Representative western blots from three independent experiments are shown. [S2F] MAC681-induced PARP1 degradation after 4 hours of treatment was prevented by calpain inhibitor PD 150606 (1-hour pre-treatment) in K-562 cells, assessed by western blot. β-Actin was used as a loading control. A representative picture from three independent experiments is shown. [S2G] Effect of MAC681 on leukemia cell line viability. The IC₅₀ value was defined as the compound concentration needed to inhibit 50 % cell viability compared to untreated controls. [S2H] Differential toxicity of MAC681 in PBMCs and K-562 cells (left panel) or RPMI1788 and K-562 cells (right panel) was estimated based on results with trypan blue exclusion assay (One-way ANOVA, Šídák's multiple comparisons test ***p ≤ 0.001, ****p ≤ 0.0001). [S2I] Colony formation assay with K-562 cells treated with MAC681 at the indicated concentrations. Quantification of total MTT signal (One-way ANOVA, Dunnett’s multiple comparisons test ***p ≤ 0.001, ****p ≤ 0.0001). [S2J] Colony formation assay with U-937, Meg-01, KBM-5, and HL-60 cells treated with MAC681 at the indicated concentrations. Images are representative of three independent experiments. Neither viability nor proliferation of the leukemia cells was affected by MAC681 before seeding the pretreated cells on methylcellulose (right panels) (One-way ANOVA, Dunnett’s multiple comparisons test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). [S2K] Effect of MAC681 on K-562 (upper panel) and U-937 (lower panel) tumor formation in zebrafish xenograft assays. Representative images from 10 fish per condition. PBS injection served as a negative control. Error bars represent the SD of at least three independent experiments. The fluorescent U-937 tumor mass was quantified and reported in the graph (One-way ANOVA, Dunnett’s multiple comparisons test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Additional file 3: Supplementary Figure 3.

[S3A] Kinetic analysis of the PARP1 protein levels in K-562 cells treated with MAC681 at 5 µM. β-actin was used as a loading control. Etoposide (VP16) and ethanol 10% (Et10%) were used as positive controls. [S3B] RT-PCR analysis of PARP1 mRNA expression at indicated time points. K-562 cells were treated with MAC681 (5 µM), 3ABA (5 mM), or their combination (One-way ANOVA, Šídák's multiple comparisons test *p ≤ 0.05, **p ≤ 0.01). [S3C] Kinetic analysis of the PARP1 protein levels in K-562 cells treated with MAC681 at 5 µM with or without cycloheximide (CHX). β-actin was used as a loading control. Mcl-1 protein expression with short turnover was used as a positive control for CHX activity. [S3D] The control docking experiment of PARP1 with 2, 3-dihydrobenzofuran-7-carboximade derivative (2US). The 2US in the complex structure of PARP1 (PDB ID: 4OQA) and the docking model are shown as red and blue stick models, respectively. Oxygen atoms are visualized in orange (upper panel). MAC681 has a similar binding mode to 2US in a complex structure of PARP1 (PDB ID: 4OQA) and PJ34 in a complex structure of PARP1 (PDB ID: 4UXB). MAC681, 2US, and PJ34 are represented as stick models in magenta, red, and yellow, respectively (lower panel). PatchDock server and AutoDock4 program were used in our docking simulation studies. [S3E] The formation of γH2AX nuclear foci in U-937, MOLM-13, MV4-11, and OCI-AML3 was quantified by flow cytometry after 4 h of MAC681 treatment (left panel) (One-way ANOVA, Dunnett’s multiple comparisons test **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Nuclear morphology was assessed by fluorescent microscopy with Hoechst staining at the same time point (right panel). [S3F] Percentage of cell death was quantified with Hoechst/PI staining in U-937 cells treated either by MAC681 (5 µM), 3ABA (1mM), VP16 (100 µM) or in a combination of 3ABA with MAC681 or with VP16 at 24 hours (upper panel). Western blot analysis of PARP1 protein cleavage in U-937 cells was determined simultaneously (lower panel). Etoposide (VP16) served as a positive control for apoptosis induction and PARP1 cleavage. [S3G] 3ABA-MAC681 adduct formation in K-652 cells (duplicate experiment). The 3ABA-MAC681-6-position adduct was detected by mass spectrometry in K-652 cells. Experimental conditions are described in the Materials and Methods section, Mass Spectrometry. MAC681-alcohol and trifluorophenol (TFP) are metabolites of MAC681. [S3H] Effect of washout of 3ABA pre-treatment on the protective effect against MAC681-mediated early and late cellular responses. Intracellular ATP content in K-562 cells pre-treated with 3ABA (5 mM) for 30 min, with or without washout, and treated for 4 hours with MAC681 (5µM) normalized to control cells was measured by CellTiter-Glo (upper left panel). Number of viable cells (upper middle panel) and viability (upper right panel) were assessed by trypan blue exclusion test after 24 h (two-way ANOVA, Fisher’s test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). The nuclear morphology of cells was visualized using Hoechst/PI staining by fluorescent microscopy (lower panel) at 24 h. [S3I] Kinetic analysis of reactive oxygen species (ROS) levels measured by flow cytometry after MAC681 (5 µM) treatment with dichlorofluorescein diacetate (DCFDA) staining (upper panel) or with redox sensor red (RSR) staining (lower panel). H₂O₂ was used as a positive control for ROS induction (double-sided unpaired t-test *p ≤ 0.05, ****p ≤ 0.0001). [S3J] 2 e^- reductase-dependent bioreductive mechanism of MAC681 activation via the iminium salt. [S3K] 2 e^- reductase-dependent bioreductive mechanism of MAC681 activation via the quinone methide. [S3L] NQO1 (upper panel) and NQO2 (lower panel) expression levels in CML patient stem cells compared to stem cells of healthy volunteers in indicated datasets (GSE5550, GSE97562+ GSE47927) (double-sided unpaired t-test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). [S3M] NQO1 (upper panel) and NQO2 (lower panel) gene expression profiles in different CML cancer cell lines from the CCLE dataset.

Additional file 4: Supplementary Figure 4.

[S4A] Dose-dependent increase in IL-8 mRNA expression induced by MAC681 at 8 h analyzed by RT PCR (One-way ANOVA, Dunnett’s multiple comparisons test **p ≤ 0.01). [S4B] Time-dependent release of HMGB1 after treatment with 5 µM of MAC681 assessed by ELISA in OCI-AML3 cells supernatants (double-sided unpaired t-test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). [S4C] Dose-dependent HMGB1 release after treatment with indicated concentrations of MAC681 assessed by ELISA in C1498 cells supernatants after 24 hours (One-way ANOVA, Dunnett’s multiple comparisons test **p ≤ 0.01, ****p ≤ 0.0001). [S4D] Quantification of cell death induced after 24 hours by MAC681 at indicated concentrations in C1498 cells analyzed by Annexin V/PI staining (One-way ANOVA, Dunnett’s multiple comparisons test ***p ≤ 0.001, ****p ≤ 0.0001). [S4E] Kinetic analysis of the weights of C57BL/6 mice (One-way ANOVA, Dunnett’s multiple comparisons test).

Additional file 5.

Additional file 6.

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Orlikova-Boyer, B., Lorant, A., Gajulapalli, S.R. et al. Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation. Biomark Res 12, 47 (2024). https://doi.org/10.1186/s40364-024-00594-w

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Received: 24 April 2024
Accepted: 27 April 2024
Published: 04 May 2024
DOI: https://doi.org/10.1186/s40364-024-00594-w

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

Abstract

Background

Methods

Results

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Introduction

Materials and methods

Computational analysis of public CML datasets

Differential expression analysis

Pathways and enrichment analysis

The translational potential of MAC681 as an antileukemic agent in vitro and in vivo

Off-target prevention of MAC681-induced cell death by the PARP inhibitor 3-aminobenzamide

MAC681-induced cell death has immunogenic potential

Synergistic potential of MAC681 in combination with asciminib in imatinib-sensitive and -resistant human leukemia

Discussion

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