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Paediatric acute myeloid leukemia (AML) is a heterogeneous hematological malignancy still heavily reliant on traditional chemotherapeutic approaches. Combination treatments have shown to be a superior approach, but their success is often hindered by side effects and different drugs' pharmacokinetics. Here, we investigated ABT-737 and Purvalanol A as a potential drug pairing for pediatric AML and described the development of CD33-targeted polymeric nanoparticles (NPs) to enable their simultaneous targeted codelivery. Separate drug encapsulation within poly(lactic- co -glycolic acid) (PLGA) NPs was optimized prior to coencapsulation of both drugs at a synergistic ratio in PEGylated PLGA NPs. The therapeutic effects of formulations were evaluated in a panel of pediatric AML cells, and dual drug-loaded NPs (dual NPs) demonstrated significantly enhanced apoptotic cell death. Moreover, conjugation to gemtuzumab resulted in improved NP binding and internalization in CD33-positive cells. Finally, CD33-targeted dual-loaded NPs showed enhanced cytotoxicity to CD33-positive AML cells via CD33-mediated targeted drug delivery.

Acute myeloid leukaemia (AML) is a lethal bone marrow neoplasm caused by genetic alterations in blood cell progenitors. Leukaemic stem cells (LSCs) are responsible for the development of AML, drug resistance and relapse. Bithionol is an old anthelmintic drug with potential antibacterial, antiviral, antifungal, anti-Alzheimer, and antitumour properties. In this work, we focused on the anti-AML LSC properties of bithionol. This compound inhibited the viability of both solid and haematological cancer cells, suppressed AML stem-like cells, and inhibited AML growth in NSG mice at a dosage of 50 mg/kg, with tolerable systemic toxicity. Bithionol significantly reduced the levels of phospho-NF-κB p65 (Ser529) and phospho-NF-κB p65 (Ser536) and nuclear NF-κB p65 translocation in AML cells, indicating that this molecule can suppress NF-κB signalling. DNA fragmentation, nuclear condensation, cell shrinkage, phosphatidylserine externalisation, loss of transmembrane mitochondrial potential, caspase-3 activation and PARP-(Asp 214) cleavage were detected in bithionol-treated AML cells, indicating the induction of apoptosis. Furthermore, this compound increased mitochondrial superoxide levels, and bithionol-induced cell death was partially prevented by cotreatment with the selective ferroptosis inhibitor ferrostatin-1, indicating the induction of ferroptosis. In addition, bithionol synergised with venetoclax in AML cells, indicating the translational potential of bithionol to enhance the effects of venetoclax in patients with AML. Taken together, these data indicate that bithionol is a potential new anti-AML drug., (© 2024. The Author(s).)

Paediatric acute myeloid leukaemia (AML) continues to present treatment challenges, as no "standard approach" exists to treat those young patients reliably and safely. Combination therapies could become a viable treatment option for treating young patients with AML, allowing multiple pathways to be targeted. Our in silico analysis of AML patients highlighted "cell death and survival" as an aberrant, potentially targetable pathway in paediatric AML patients. Therefore, we aimed to identify novel combination therapies to target apoptosis. Our apoptotic drug screening resulted in the identification of one potential "novel" drug pairing, comprising the Bcl-2 inhibitor ABT-737 combined with the CDK inhibitor Purvalanol-A, as well as one triple combination of ABT-737 + AKT inhibitor + SU9516, which showed significant synergism in a series of paediatric AML cell lines. Using a phosphoproteomic approach to understand the apoptotic mechanism involved, proteins related to apoptotic cell death and cell survival were represented, in agreement with further results showing differentially expressed apoptotic proteins and their phosphorylated forms among combination treatments compared to single-agent treated cells such upregulation of BAX and its phosphorylated form (Thr167), dephosphorylation of BAD (Ser 112), and downregulation of MCL-1 and its phosphorylated form (Ser159/Thr 163). Total levels of Bcl-2 were decreased but correlated with increased levels of phosphorylated Bcl-2, which was consistent with our phosphoproteomic analysis predictions. Bcl-2 phosphorylation was regulated by extracellular-signal-regulated kinase (ERK) but not PP2A phosphatase. Although the mechanism linking to Bcl-2 phosphorylation remains to be determined, our findings provide first-hand insights on potential novel combination treatments for AML.

Paediatric acute myeloid leukaemia (AML) is a heterogeneous disease characterised by the malignant transformation of myeloid precursor cells with impaired differentiation. Standard therapy for paediatric AML has remained largely unchanged for over four decades and, combined with inadequate understanding of the biology of paediatric AML, has limited the progress of targeted therapies in this cohort. In recent years, the search for novel targets for the treatment of paediatric AML has accelerated in parallel with advanced genomic technologies which explore the mutational and transcriptional landscape of this disease. Exploiting the large combinatorial space of existing drugs provides an untapped resource for the identification of potential combination therapies for the treatment of paediatric AML. We have previously designed a multiplex screening strategy known as Multiplex Screening for Interacting Compounds in AML (MuSICAL); using an algorithm designed in-house, we screened all pairings of 384 FDA-approved compounds in less than 4000 wells by pooling drugs into 10 compounds per well. This approach maximised the probability of identifying new compound combinations with therapeutic potential while minimising cost, replication and redundancy. This screening strategy identified the triple combination of glimepiride, a sulfonylurea; pancuronium dibromide, a neuromuscular blocking agent; and vinblastine sulfate, a vinca alkaloid, as a potential therapy for paediatric AML. We envision that this approach can be used for a variety of disease-relevant screens allowing the efficient repurposing of drugs that can be rapidly moved into the clinic.