Background:

The Lysine Methyltransferase 2A (KMT2A) gene can partner with over 60 fusion partners. KMT2A mediates chromatin methylation at histone H3 lysine 4 (H3K4), which is associated with epigenetic transcriptional activation. KMT2A fusions are present in high-risk acute leukemias affecting lymphoid and myeloid cell lineages. The most frequent Acute Lymphoblastic Leukemia (ALL) fusion partner gene is AFF1 (KMT2A-AFF1), and the most common Acute Myeloid Leukemia (AML) partner is MLLT3 (KMT2A-MLLT3). During disease progression, lineage drift or switching from lymphoid to myeloid can occur and may include presence of biphenotypic markers. The fusions confer a poor prognosis and occur in 70% to 80% of infants with ALL. The rearrangement of KMT2A (KMT2Ar) is considered a driver mutation. A single agent drug repositioning screen and an iterative drug combination synergy screen were conducted to identify new therapies and combinations that are more effective at eliminating these therapy-resistant AMLs.

Methods:

We employed a drug repositioning strategy using a library of 3,832 drugs, comprising 66% FDA approved drugs and 33% that have passed Phase 1 clinical trials, to identify therapies that are effective against KMT2Ar AMLs. We tested a diverse range of models, including KMT2A rearranged cell lines (n=3) and KMT2A non-rearranged patient derived AMLs (n=3), generating data from 150,000 wells. To further evaluate a single-agent lead that ranked in the top 1% of cytotoxic compounds, a secondary screen was conducted. This involved testing SN-38 in combination with the same 3,832 drugs against the KMT2A-MLLT3 cell line MOLM-13 to assess synergistic potential.

Results:

High Throughput Drug Screening identified the top five most active FDA-approved drugs across 6 AML cell lines: 1) SN-38 (a topoisomerase I inhibitor and the active metabolite of irinotecan), 2) Triplotide (an NF-κB inhibitor), 3) Mitoxantrone (a topoisomerase II inhibitor), 4) Idarubicin (a topoisomerase II inhibitor), and 5) Bortezomib (a proteasome inhibitor). Topoisomerase I inhibitors like camptothecin and its derivatives, such as irinotecan, demonstrated significant effectiveness at low nanomolar concentrations across all AML cell lines. Drugs that exhibited greater selectivity, meaning greater responses in KMT2Ar cell lines, included: 1) Ingenol Mebutate (a PKC inhibitor), 2) a BET bromodomain inhibitor, 3) MIK665 (an MCL1 inhibitor), 4) Prexasertib (a CHK inhibitor), and 5) Crenolanib (an FLT3/PDGFR inhibitor). We focused our secondary screening efforts on SN-38, identified as the most potent overall agent in the initial screen of all AML samples. A secondary screen was then conducted using the 3,832-drug library to identify the most effective combinations against KMT2Ar AML. Our assessment of synergistic effects showed that SN-38 displayed significant synergy with PARP and BCL-2 pathway inhibitors in the KMT2A-MLLT3 MOLM-13 AML cell line. Overall, our data indicate that SN-38 possesses highly potent anti-leukemic activity against pediatric KMT2Ar AMLs. Notably, SN-38 has reported efficacy as a single agent in patient-derived xenograft mouse models of KMT2A-AFF1 ALL (Kerstjens et al. Biomedicines 2021).

Conclusion:

SN-38, the active metabolite of irinotecan, demonstrates potential as an anti-leukemic agent in AML. Its potent inhibitory effects, especially in KMT2A-rearranged (KMT2Ar) leukemias, make SN-38 a compelling candidate for further investigation and potential clinical application. Moreover, the observed synergy with PARP and BCL-2 inhibitors reveals strategies to overcome AML resistance mechanisms to SN-38. However, rigorous preclinical and clinical studies are essential to validate its efficacy, safety, and optimal dosing regimens.

Disclosures

No relevant conflicts of interest to declare.

Off Label Disclosure:

SN-38 has been used in clinical trials for the treatment of various cancers, including advanced solid tumors, small cell lung cancer, metastatic colorectal cancer, and triple-negative breast cancer

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2024
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