Acute myeloid leukemia (AML) is a hematologic malignancy that represents ~20% of pediatric leukemias, but accounts for most of the disease-related mortality in children. Given the genetic heterogeneity of pediatric AML that impacts patient survival rate and relapse, a single treatment strategy does not exist for all AML subtypes. This underlines the urgent need for novel targeted (genotype specific) therapies. Through a high-throughput drug screen comparing the growth inhibition of 37 AML samples and normal cord blood stem cells (CB CD34+) treated with approximately 11,000 compounds, we identified compound 280 which is structurally related to a previously reported proteasome inhibitor.

In collaboration with the Drug Discovery Unit at the Institute for Research in Immunology and Cancer (IRIC), we synthesized and characterized over 80 analogs of compound 280. We determined the half-maximal inhibitory concentrations (IC50) for these analogs in cell viability assays using three AML cell lines and conducted purified proteasome inhibition assays to further evaluate their proteasome inhibitory activities. To assess the potential mechanisms of action of these analogs, we performed a CRISPR chemogenomic screen in the B-ALL cell line NALM-6 using the top two analogs and Bortezomib (BTZ). To evaluate the behavior of these compounds in vivo, we have conducted pharmacokinetic (PK) studies, as well as metabolic stability in animal models.

Several analogs exhibited lower proteasome inhibition compared to BTZ based on the enzymatic IC50 values, but demonstrated greater efficacy in inhibiting AML cell growth, as indicated by cell-based IC50 values. CRISPR chemogenomic analysis confirmed that both our analogs and BTZ target the proteasome. However, the loss of genes linked to resistance mechanisms, including those associated with drug efflux, increased cell sensitivity to the analogs, but did not alter sensitivity to BTZ. This finding suggests that while both BTZ and our novel compounds target the proteasome, they likely rely on different molecular mechanisms of resistance. PK studies indicated that the selected analogs, when administered intraperitoneally (IP), showed favorable stability, prolonged systemic exposure, and appropriate PK parameters, supporting their potential for further development.

Our novel proteasome inhibitor shows promising potential for clinical application in pediatric AML. By understanding how compound 280 and its analogs impact AML cell growth and resistance mechanisms, we aim to develop targeted therapies tailored to specific genetic alterations in AML patients. Furthermore, these therapies may extend their applicability to other hematologic disorders and solid tumors, addressing a broader spectrum of cancers.