Autophagy is an evolutionarily conserved lysosomalmediated degradation process that facilitates the turnover of organelles and selected long-lived proteins. Aberrant autophagy contributes to therapeutic resistance and malignant progression by generating alternative sources of metabolic fuel to maintain cell survival under stress conditions, including those imposed by hypoxia, radiation, chemotherapy, and targeted agents [1]. The FDA approved antimalarial drug hydroxychloroquine (HCQ) is known to inhibit autophagy through the disruption of lysosomal function. Based on this aspect of its mechanism of action, tremendous efforts were undertaken to repurpose it for cancer therapy over the last decade. Although HCQ has been combined with an array of other anticancer regimens in clinical trials, these studies have predominantly reported modest clinical benefit characterized by partial responses and stable disease, but few sustained complete responses [2, 3]. Despite the initial subdued clinical results reported from these trials, there is maintained enthusiasm for autophagy inhibition as an anticancer approach for two overarching reasons. First, it remains unclear if the maximum tolerated dose of HCQ when used in combination with other anticancer agents is sufficient to completely inhibit autophagy in tumors. The pharmacodynamic (PD) studies conducted in support of most of these early phase HCQ combination trials were not powered to test this in a definitive manner. Residual autophagic activity may have been a factor that limited efficacy in these studies. Second, despite its prevalent use as an autophagy inhibitor, the reality is that HCQ is a very old drug that was not designed to inhibit autophagy. Its continued use for this purpose is driven by the lack of better alternatives. New autophagy inhibitors with increased potency and more favorable therapeutic indices are clearly needed to definitively determine the efficacy of this approach, but none have been tested in humans to date.

We recently generated a series of novel dimeric compounds containing modified core elements of HCQ and the antischistosomal drug lucanthone with the goal of developing new lysosomal autophagy inhibitors with superior potency, tolerability, and anticancer efficacy [4, 5]. Initial preclinical studies identified ROC-325 as a lead agent for further investigation, as it demonstrated approximately tenfold greater potency than HCQ, and is an orally bioavailable compound. We selected acute myeloid leukemia (AML) as a priority malignancy for further investigation based on the high sensitivity of AML cells to ROC-325 in early screens and the frequent aberrations in the autophagy interactome in patients with myeloid neoplasms that we recently reported [6]. We first quantified the effects of ROC-325 on the hallmark features of autophagy inhibition in AML cells. Transmission electron microscopy analyses demonstrated that treatment of AML cells with ROC-325 triggered the accumulation of autophagosomes with undegraded cargo (Fig. 1a). The majority of lysosomal proteases that control the autophagy-mediated degradation of proteins and organelles require a strict acidic pH microenvironment for their enzymatic activity. Based on this, lysosomal