Nonclinical evaluation of APL-4098, a novel GCN2 kinase inhibitor, reveals potent anti-leukemic effects through mitochondrial stress induction and synergy with venetoclax, targeting AML blasts and LSCs

Abstract Background: Despite recent progress in acute myeloid leukemia (AML) therapy, long-term outcomes remain dismal, primarily due to the persistence of leukemia stem cells (LSCs) that drive progression, relapse and drug resistance. GCN2 (general control nondepressible 2) is an evolutionarily conserved kinase and a pivotal regulator of the integrated stress response (ISR) activated in response to amino acid scarcity and other extrinsic and intrinsic stresses. Activation of GCN2 phosphorylates translation initiation factor eIF2α resulting in the attenuation of global protein synthesis and a transcriptional rescue program. GCN2 activation in response to amino acid deprivation is a mechanism by which tumor cells can survive under nutrient stress. GCN2 is therefore an interesting new target for therapeutic development and here we describe the nonclinical anti-leukemic activity of the novel small molecule GCN2 inhibitor APL-4098 and elucidate its mechanism of action. Methods: GCN2 inhibition by APL-4098 was confirmed using a biochemical Lanthascreen assay and a cell-based homogeneous time resolved fluorescence (HTRF) assay measuring eIF2α phosphorylation following activation with Borrelidin. Kinase selectivity was determined using KinomeScan®. Inhibition of leukemia cell viability and induction of cell death was investigated ex vivo in AML patient samples using Cell Titer Glo and Annexin V staining. Effects of APL-4098 on AML LSCs and blasts were investigated in vivo using AML cell line-derived (CDX) and patient-derived (PDX) mouse models using APL-4098 as a single agent and in combination with the BCL-2 inhibitor venetoclax. RNA sequencing of tumor samples and patient-derived AML cells was conducted to explore the mechanism of action of APL-4098. RESULTS: APL-4098 potently inhibited GCN2 activity, with a Ki of 4.4 nM as measured by the biochemical Lanthascreen assay. Furthermore, in cell-based assays, following GCN2 activation with Borrelidin, APL-4098 inhibited eIF2a phosphorylation with an IC50 of 50.8nM. The KinomeScan® confirmed that APL-4098 was highly selective, with minimal off-target activity. 30 primary AML patient samples were exposed to increasing doses of APL-4098. Of these, 21 samples showed a reduction in cell viability of more than 50%, and 6 samples exhibited a reduction in viability between 25% and 50%. Dose-dependent induction of apoptosis was confirmed in 6 additional patient samples. In 3 of these samples, the effect of APL-4098 was evaluated on the LSCs enriched compartment (CD34+/CD38-) and a significant reduction in this subpopulation was observed in all samples. APL-4098 in vivo efficacy was first studied in a MOLM-16 AML CDX model. Mice were treated once daily with APL-4098 at doses ranging between 0.5mg/kg and 5 mg/kg daily for 14 days. APL-4098 inhibited tumor growth in a dose-dependent manner, with a tumor growth inhibition (TGI) between -11.56% (0.5 mg/kg) to 98.7% (5 mg/kg). In an AML PDX model, once daily treatment with 15 mg/kg APL-4098 as a single agent resulted in more than 50% reduction of the LSC-enriched compartment. Co-administration of APL-4098 with venetoclax resulted in a significant synergistic effect on total blast count and LSCs. RNA sequencing of AML patient cells treated ex vivo with 1 µM APL-4098 and of AML cells collected from the CDX model showed a rapid and widespread impact of APL-4098. Gene Set Enrichment Analysis revealed profound effects on cellular energy metabolism, with a significant downregulation of genes involved in oxidative phosphorylation, mitochondrial transport and mitochondrial translation. In parallel, we observed a transcriptional response indicative of a mitochondrial stress response and a mitochondrial unfolded protein response. Conclusion: APL-4098 is a novel, potent, selective, ATP-competitive GCN2 kinase inhibitor that disrupts mitochondrial processes and targets LCSs in nonclinical AML models as a monotherapy and in combination with venetoclax. A Phase 1/2 clinical study of APL-4098 in R/R AML and MDS/AML is ongoing (NCT06372717).