Combined autophagy and proteasome inhibition: a phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma

Abstract

The efficacy of proteasome inhibition for myeloma is limited by therapeutic resistance, which may be mediated by activation of the autophagy pathway as an alternative mechanism of protein degradation. Preclinical studies demonstrate that autophagy inhibition with hydroxychloroquine augments the antimyeloma efficacy of the proteasome inhibitor bortezomib. We conducted a phase I trial combining bortezomib and hydroxychloroquine for relapsed or refractory myeloma. We enrolled 25 patients, including 11 (44%) refractory to prior bortezomib. No protocol-defined dose-limiting toxicities occurred, and we identified a recommended phase 2 dose of hydroxychloroquine 600 mg twice daily with standard doses of bortezomib, at which we observed dose-related gastrointestinal toxicity and cytopenias. Of 22 patients evaluable for response, 3 (14%) had very good partial responses, 3 (14%) had minor responses, and 10 (45%) had a period of stable disease. Electron micrographs of bone marrow plasma cells collected at baseline, after a hydroxychloroquine run-in, and after combined therapy showed therapy-associated increases in autophagic vacuoles, consistent with the combined effects of increased trafficking of misfolded proteins to autophagic vacuoles and inhibition of their degradative capacity. Combined targeting of proteasomal and autophagic protein degradation using bortezomib and hydroxychloroquine is therefore feasible and a potentially useful strategy for improving outcomes in myeloma therapy.

Keywords: autophagy; myeloma; proteasome.

Figure 1. Schema of study treatment and acquisition of correlative samples. Patients started a 2-wk run-in of single-agent hydroxychloroquine (HCQ), which they continued during 3 wk cycles of standard bortezomib (B) on d 1, 4, 8, and 11. Solid arrows indicate time points at which samples were obtained for all patients; this included peripheral blood mononuclear cells obtained at baseline, on d 1 and 8 of cycle 1, and on d 1 of cycle 2, as well as bone marrow samples obtained at baseline and on d 1 of cycle 1. The hatched arrows indicate that the bone marrow samples obtained on combined HCQ and bortezomib were either on d 5 of cycle 1 (the final 3 patients) or d 1 of cycle 2 (all previous patients).

Figure 2. Therapy-associated autophagy modulation in myeloma cells from a patient treated with hydroxychloroquine 400 mg daily and standard bortezomib. (A) Representative electron micrographs of CD138-selected bone marrow plasma cells. Red arrows indicate AVs; orange arrows indicate mitochondria. Scale bar: 2 µm. Samples were obtained from a single patient (patient 13, on dose cohort 4) prior to treatment (baseline), after the 2-wk HCQ run-in on d 1 of cycle 1, and prior to therapy on d 1 of cycle 2. (B) Quantification of AVs in bone marrow plasma cells from patient 13 obtained at baseline, d 1 of cycle 1 (C1D1), and d 1 of cycle 2 (C2D1). Vacuole counts from 2 assessors blinded to patient and time point were averaged, with error bars reflecting the standard error of measurement across 25 cells per sample. The treatment schema shows the timing of administration of daily oral hydroxychloroquine (HCQ) and intravenous bortezomib (B). (C) Immunoblotting of bone marrow plasma cells from patient 13 at the same time points.

Figure 3. Therapy-associated autophagy modulation in myeloma and peripheral blood mononuclear cells from patients treated with hydroxychloroquine (HCQ) and bortezomib. Shown are mean autophagic vacuole counts in (A) bone marrow plasma cells and (B) PBMCs sampled during therapy. The bone marrow results were taken at baseline, on d 1 of cycle 1 (C1D1, after a 2-wk HCQ run-in), and either on d 5 of cycle 1 (C1D5, ~24 h after the d 4 bortezomib dose) or on d 1 of cycle 2 (C2D1, ~10 d after the d 11 bortezomib dose). Peripheral blood samples were obtained at baseline, on C1D1, on d 8 of cycle 1 (C1D8), and on C2D1. P values are shown for comparisons across all time points, and asterisks indicate time points that are significantly (P < 0.05) different from baseline. The treatment schema below each panel shows the timing of administration of daily oral hydroxychloroquine (HCQ) and intravenous bortezomib (B). (C) Correlation between autophagic vacuole counts in peripheral blood mononuclear cells and bone marrow plasma cells for all time points at which patients had both samples obtained at the same time. Correlation coefficients were not statistically significant at baseline (0.34, P = 0.21), C1D1 (0.10, P = 0.77), or C2D1 (0.65, P = 0.18).

Figure 4. Correlation of predicted and observed hydroxychloroquine (HCQ) levels. Shown are observed HCQ whole blood concentrations vs. individual predicted concentrations from a population pharmacokinetic model in patients treated with HCQ and bortezomib.

Figure 5. Hydroxychloroquine (HCQ) levels in patients receiving HCQ and bortezomib for myeloma. Shown are steady-state HCQ whole blood concentrations, by average daily HCQ dose. Steady-state HCQ concentrations are estimated from a population pharmacokinetic model. The graph shows the median (solid horizontal line), mean (dashed horizontal line), 25th–75th percentiles (box), and 5th–95th percentiles (whiskers).