Chromomycin A2 exhibits antileukemic effects by coordinated disruption of survival pathways Free

New therapies for acute leukemias are urgently needed due to the high relapse rates and the frequent development of resistance to conventional treatments. The identification of specific molecular targets has enabled the development of more effective and less toxic strategies, with the potential to improve both survival and quality of life. Furthermore, innovative therapeutic approaches may benefit patient subgroups with poor prognosis and limited treatment options. In this context, the chromomycin class has emerged as a promising therapeutic candidate, owing to its ability to interfere with DNA structure and modulate key cellular survival pathways. Chromomycins bind to GC-rich regions of DNA, intercalate into the helix, and inhibit RNA polymerase activity, thereby blocking RNA synthesis and gene expression. Among these compounds, chromomycin A2 remains underexplored. It has demonstrated antitumor activity in melanoma and gastric adenocarcinoma models, but its effects on hematologic malignancies have not been investigated to date.

In the present study, we evaluated the antineoplastic effects of chromomycin A2 across a broad panel of myeloid and lymphoid malignancies, as well as the cellular and molecular events triggered by the compound. A total of 22 leukemia-derived cell lines were analyzed, including myeloid (n = 13) and lymphoid (n = 9) models, with subsets with acquired resistant to venetoclax (n = 2), quizartinib (n = 1), and ATRA (n = 1). Cell viability was assessed by MTT assay, apoptosis by annexin V/PI staining and flow cytometry, and autonomous clonal growth in cytokine- and growth factor-free methylcellulose-based semi-solid medium. Additionally, ex vivo experiments were conducted using primary cells from adult patients with acute myeloid leukemia (AML; n = 3) and acute lymphoblastic leukemia (ALL; n = 3). Molecular analyses included PCR arrays targeting 22 key genes involved in autophagy, cell cycle progression, apoptosis, and DNA damage, as well as Western blotting. Statistical comparisons were performed using ANOVA followed by Bonferroni post hoc test or Student's t-test, with p < 0.05 considered significant.

Chromomycin A2 significantly reduced the viability of all leukemia-derived cell lines in a dose-dependent manner, with IC₅₀ values ranging from 0.40 to 4.42 nM. Acquired resistant leukemia models showed slightly higher IC₅₀ values compared to parental lines, yet remained within the sensitivity range. The effects on viability reduction of chromomycin A2 were time-dependent and associated with concentration-dependent induction of apoptosis. The compound completely abolished autonomous clonal growth of both AML and ALL cell lines at concentrations ≥ 5 nM. At the molecular level, early treatment with chromomycin A2 induced autophagic markers (LC3B-II accumulation and SQSTM1/p62 degradation), likely reflecting transcriptional suppression and growth factor-related signal reduction. At later time points, apoptotic markers (cleaved PARP1) and DNA damage indicators (γH2AX) became prominent. Gene expression analyses supported this sequence, showing early upregulation of autophagy- and cell cycle arrest–related genes (6 h), followed by induction of apoptosis-related genes (12 h). Network and gene ontology enrichment analyses highlighted chromomycin A2 activity in macroautophagy, mitochondrial apoptotic processes, and G1 DNA damage checkpoint pathways (FDR < 0.001). In ex vivo assays, chromomycin A2 showed IC₅₀ values ranging from 1.6 to 31.8 nM and efficacy levels of 81-92% in primary AML and ALL samples.

In conclusion, chromomycin A2 exhibited potent antileukemic activity in both established cell lines and patient-derived ex vivo models, including subtypes with acquired resistance to conventional therapies. Its effects were dose- and time-dependent, associated with the sequential induction of autophagy, apoptosis, and DNA damage, consistent with a multitarget mechanism of action. The compound also effectively suppressed autonomous clonal growth, highlighting its potential to impair leukemic stem/progenitor cell function. Taken together, these findings support the therapeutic potential of chromomycin A2 in hematologic malignancies and provide a strong rationale for further preclinical studies. Supported by FAPESP, CAPES, and CNPq.