Development of ROC-325: A Novel Small Molecule Inhibitor of Autophagy with Promising Anti-Leukemic Activity

Autophagy contributes to therapeutic resistance and malignant progression by generating alternative metabolic fuel to maintain cell survival under stress conditions including those imposed by hypoxia, radiation, chemotherapy, and targeted agents. The FDA approved anti-malarial drug hydroxychloroquine (HCQ) inhibits autophagy through the disruption of lysosomal function. Robust efforts to repurpose HCQ for cancer therapy based on these properties stimulated numerous clinical trials where HCQ was combined with an array of other anticancer regimens and ultimately produced modest clinical activity. However, it was unclear if the maximum tolerated dose of HCQ was sufficient to completely inhibit autophagy in tumors. New autophagy inhibitors with increased potency and more favorable therapeutic indices are clearly needed to rigorously investigate the potential benefit of this approach.

Polyvalent molecules can yield nonlinear, multifold potency against their respective targets compared to their corresponding monomers. We generated a series of novel dimeric compounds containing modified core elements of HCQ, CQ and the anti-schistosomal drug lucanthone with the goal of developing new autophagy inhibitors with superior potency, tolerability, and anticancer efficacy. Initial screens that tested for drug-induced increased expression of p62, a protein that is specifically turned over by autophagy, and anticancer activity identified ROC-325 as a lead agent. The structure of ROC-325 was confirmed by NMR and MS analyses. Direct comparison of the in vitro activity of ROC-325 and HCQ in 13 different genetically and histologically diverse human cancer cell lines demonstrated that ROC-325 was approximately 10-fold more potent than HCQ based on IC₅₀ analyses. Acute myeloid leukemia (AML) was selected as a primary malignancy for intensive investigation based on the high sensitivity of FLT3-ITD+ MV4-11 cells to this agent in preliminary studies. Transmission electron microscopy analyses demonstrated that treatment with ROC-325 triggered all of the hallmark features of autophagy inhibition including the accumulation of autophagosomes with undegraded cargo, an increase in lysosomal membrane permeability, deacidification of lysosomes, and elevated LC3B, p62, and cathepsin D expression. Bafilomycin A1 clamp experiments showed that ROC-325 potently inhibited autophagic flux. Genetic impairment of two different genes that are essential for functional autophagy, ATG5 and ATG7, using lentiviral shRNA approaches significantly diminished the anticancer effects of ROC-325, thus indicating that autophagy inhibition is a key component of its anticancer mechanism of action.RNA sequencing and gene level analyses demonstrated that treatment with ROC-325 (1 µM) in MV4-11 cells altered the levels of autophagy-dependent degradation pathways while preserving protein synthesis through the upregulation of post-translational ribosomal, methylation, and splicing components.

In vitro treatment of a panel of human AML cell lines and normal human bone marrow progenitors demonstrated that ROC-325 diminished AML cell viability (IC50 range 0.7-2.2 µM), antagonized clonogenic survival, and induced apoptosis in a manner that was therapeutically selective. Analysis of primary blasts from patients with AML showed that its activity was not significantly affected by adverse cytogenetics or multi-drug resistance due to relapsed/refractory clinical status. Oral administration of 50 mg/kg ROC-325 (QDx5) to mice bearing disseminated MV4-11 human AML xenografts significantly increased lifespan (P<0.05) and enhanced the efficacy of azacitidine (5 mg/kg IV, 2X per week). ROC-325 was well tolerated and no notable toxicities were observed other than a modest, non-significant reversible reduction in mean body weight. Immunohistochemical analysis of specimens collected from animals treated with ROC-325 demonstrated significant increases in the autophagic markers LC3B and p62 and apoptotic blasts. Our data demonstrate that ROC-325 is a novel orally available autophagy inhibitor that is well tolerated, significantly more potent than HCQ, and has promising anti-AML activity. These findings support further investigation of the safety and efficacy of ROC-325 as a novel agent for the treatment of AML and other disorders where lysosomal activity contributes to disease pathogenesis.