Targeting Autophagy in Cancer: Update on Clinical Trials and Novel Inhibitors

A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.

Preclinical studies indicate autophagy inhibition with hydroxychloroquine (HCQ) can augment the efficacy of DNA-damaging therapy. The primary objective of this trial was to determine the maximum tolerated dose (MTD) and efficacy of HCQ in combination with radiation therapy (RT) and temozolomide (TMZ) for newly diagnosed glioblastoma (GB). A 3 + 3 phase I trial design followed by a noncomparative phase II study was conducted in GB patients after initial resection. Patients received HCQ (200 to 800 mg oral daily) with RT and concurrent and adjuvant TMZ. Quantitative electron microscopy and immunoblotting were used to assess changes in autophagic vacuoles (AVs) in peripheral blood mononuclear cells (PBMC). Population pharmacokinetic (PK) modeling enabled PK-pharmacodynamic correlations. Sixteen phase I subjects were evaluable for dose-limiting toxicities. At 800 mg HCQ/d, 3/3 subjects experienced Grade 3 and 4 neutropenia and thrombocytopenia, 1 with sepsis. HCQ 600 mg/d was found to be the MTD in this combination. The phase II cohort (n = 76) had a median survival of 15.6 mos with survival rates at 12, 18, and 24 mo of 70%, 36%, and 25%. PK analysis indicated dose-proportional exposure for HCQ. Significant therapy-associated increases in AV and LC3-II were observed in PBMC and correlated with higher HCQ exposure. These data establish that autophagy inhibition is achievable with HCQ, but dose-limiting toxicity prevented escalation to higher doses of HCQ. At HCQ 600 mg/d, autophagy inhibition was not consistently achieved in patients treated with this regimen, and no significant improvement in overall survival was observed. Therefore, a definitive test of the role of autophagy inhibition in the adjuvant setting for glioma patients awaits the development of lower-toxicity compounds that can achieve more consistent inhibition of autophagy than HCQ.

The combination of temsirolimus (TEM), an MTOR inhibitor, and hydroxychloroquine (HCQ), an autophagy inhibitor, augments cell death in preclinical models. This phase 1 dose-escalation study evaluated the maximum tolerated dose (MTD), safety, preliminary activity, pharmacokinetics, and pharmacodynamics of HCQ in combination with TEM in cancer patients. In the dose escalation portion, 27 patients with advanced solid malignancies were enrolled, followed by a cohort expansion at the top dose level in 12 patients with metastatic melanoma. The combination of HCQ and TEM was well tolerated, and grade 3 or 4 toxicity was limited to anorexia (7%), fatigue (7%), and nausea (7%). An MTD was not reached for HCQ, and the recommended phase II dose was HCQ 600 mg twice daily in combination with TEM 25 mg weekly. Other common grade 1 or 2 toxicities included fatigue, anorexia, nausea, stomatitis, rash, and weight loss. No responses were observed; however, 14/21 (67%) patients in the dose escalation and 14/19 (74%) patients with melanoma achieved stable disease. The median progression-free survival in 13 melanoma patients treated with HCQ 1200mg/d in combination with TEM was 3.5 mo. Novel 18-fluorodeoxyglucose positron emission tomography (FDG-PET) measurements predicted clinical outcome and provided further evidence that the addition of HCQ to TEM produced metabolic stress on tumors in patients that experienced clinical benefit. Pharmacodynamic evidence of autophagy inhibition was evident in serial PBMC and tumor biopsies only in patients treated with 1200 mg daily HCQ. This study indicates that TEM and HCQ is safe and tolerable, modulates autophagy in patients, and has significant antitumor activity. Further studies combining MTOR and autophagy inhibitors in cancer patients are warranted.