Background: The antiangiogenic tyrosine kinase inhibitor regorafenib provides a survival benefit in patients with previously treated metastatic colorectal cancer. Antiangiogenic therapy causes hypoxic stress within tumor cells, which activate autophagy as a survival mechanism. Entinostat, a histone deacetylase (HDAC) inhibitor, increases dependence on autophagy through epigenetic mechanisms. Hydroxychloroquine (HCQ) blocks autophagy by blunting lysosomal acidification and is synergistic with antiangiogenic therapies. We hypothesized that HCQ and entinostat would be tolerable with regorafenib and potentiate the antitumor response. Methods: This was a 3+3 phase I trial to find the recommended phase II dose (RP2D) of HCQ and entinostat with regorafenib in patients with metastatic colorectal cancer previously treated with a fluoropyrimidine, oxaliplatin, and irinotecan. No prior regorafenib or HDAC inhibitor therapy was permitted. Regorafenib was dosed at 160mg daily on days 1-21 of 28-day cycles, with provision to lower the starting dose to 80mg if toxicity was excessive. Entinostat was dosed at 3mg weekly in dose level 1 and at 5mg weekly in dose levels 2 and 3 while HCQ was dosed at 200mg qAM and 400mg qPM in dose levels 1 and 2 and at 600mg BID at dose level 3. Expansion was planned at the RP2D with a primary endpoint of objective response rate. Results: Twenty-eight patients were screened, and 20 patients were enrolled from November 2017 to January 2020. Six patients were treated at dose level 1 with no dose-limiting toxicity. The starting regorafenib dose was reduced to 80mg after 3 patients discontinued therapy early due to fatigue or rash due to regorafenib. At dose level 2, 7 patients were enrolled to achieve 6 evaluable patients. One DLT (G3 fatigue) was noted and one patient withdrew consent after 14 days due to fever and tumor pain flare possibly related to treatment. Six patients enrolled at dose level 3; no DLTs were seen. One additional patient received HCQ 400mg BID instead of 600mg BID due to a clerical error. Weight loss (60%), fatigue (50%), and anorexia (50%) were the most common toxicities. Thirteen grade 3 toxicities were noted, with rash (15%), fatigue (10%), and alkaline phosphatase elevation (10%) the most common. No grade 4 toxicities were observed. Seven patients discontinued therapy early due to toxicity. Nearly all patients experienced rapid weight loss, with a range of 1.5 lbs to 27.1 lbs and a median weight loss of 9.5 lbs at two weeks. No objective responses were observed. The median PFS was 1.8 months, the median OS was 5.2 months, and no patient remained on study longer than 4 months. Expansion was not pursued due to toxicity and lack of efficacy. Conclusions: The combination of regorafenib, HCQ, and entinostat was poorly tolerated without evident activity in metastatic colorectal cancer. The substantial weight loss suggests a potential adverse metabolic interaction between these drugs. Clinical trial information: NCT03215264