Molecular targeting of the UDP-glucuronosyltransferase enzymes in high-eukaryotic translation initiation factor 4E refractory/relapsed acute myeloid leukemia patients: a randomized phase II trial of vismodegib, ribavirin with or without decitabine

Abstract

Drug resistance underpins poor outcomes in many malignancies including refractory and relapsed acute myeloid leukemia (R/R AML). Glucuronidation is a common mechanism of drug inactivation impacting many AML therapies, e.g., cytarabine, decitabine, azacytidine and venetoclax. In AML cells, the capacity for glucuronidation arises from increased production of the UDP-glucuronosyltransferase 1A (UGT1A) enzymes. UGT1A elevation was first observed in AML patients who relapsed after response to ribavirin, a drug used to target the eukaryotic translation initiation factor eIF4E, and subsequently in patients who relapsed on cytarabine. UGT1A elevation resulted from increased expression of the sonic-hedgehog transcription factor GLI1. Vismodegib inhibited GLI1, decreased UGT1A levels, reduced glucuronidation of ribavirin and cytarabine, and re-sensitized cells to these drugs. Here, we examined if UGT1A protein levels, and thus glucuronidation activity, were targetable in humans and if this corresponded to clinical response. We conducted a phase II trial using vismodegib with ribavirin, with or without decitabine, in largely heavily pre-treated patients with high-eIF4E AML. Pre-therapy molecular assessment of patients' blasts indicated highly elevated UGT1A levels relative to healthy volunteers. Among patients with partial response, blast response or prolonged stable disease, vismodegib reduced UGT1A levels, which corresponded to effective targeting of eIF4E by ribavirin. In all, our studies are the first to demonstrate that UGT1A protein, and thus glucuronidation, are targetable in humans. These studies pave the way for the development of therapies that impair glucuronidation, one of the most common drug deactivation modalities. Clinicaltrials.gov: NCT02073838.

Figure 1.

Schematic of forms of drug resistance relevant to this trial. Glucuronidation of ribavirin (Rib) and decitabine (Dec) is indicated by red boxes; the ENT1 transporter is depicted as the purple channel. While not shown, drug resistance can also occur via the simultaneous loss of ENT1 and elevated UGT1A (and thus increased glucuronidation). Model created using Biorender.

Figure 2.

Characterization of UGT1A levels in cells and primary acute myeloid leukemia specimens. (A) UGT1A antibody purification is described in the Online Supplementary Methods. The resulting antibody revealed a single band when monitoring endogenous UGT1A in liver HepG2 cells on the western blot with reduction for pan-UGT1A RNAi versus luciferase RNAi controls. Actin blot is provided for loading. Pan-UGT1A antibody purified in (A) recognized elevated UGT1A in THP-1 cells over-expressing GLI1 as assessed both by western blot (B) and confocal microscopy (C). In addition, UGT1A levels are reduced by vismodegib in these cells (B). (D) UGT1A are elevated in AML patients, including some treatment naïve patients, relative to healthy volunteers. UGT1A levels were observed to be elevated in all the AML patient specimens examined including 3/4 treatment naïve AML patients relative to healthy volunteers. Scale bar: 10 µm.

Figure 3.

Molecular response of eIF4E and UGT1A for patient B-004 who achieved a blast response. (A) Samples for completed treatment cycles (CT) 1 to 10 (1CT-10CT) were collected at the end of each cycle; the patient had a 28-day treatment interruption after 9CT. Percentage of bone marrow blasts is shown (in black). Changes in protein expression of eIF4E (green) and UGT1A (red) in AML blasts isolated by FACS and stained as described in the Online Supplementary Appendix. Graph generated using GraphPad Prism 7. (B) Representative confocal micrographs of blasts used for quantification in (A). eIF4E (green), UGT1A (red) and DAPI (blue) in sorted bone marrow blasts are shown. Note also that a higher fraction of eIF4E is in the nucleus before treatment (BT) and at end of treatment (EOT) than during response, as observed previously in patients treated with ribavirin.^, Dapi provided as a nuclear marker. Scale bars: 10 µm.

Figure 4.

UGT1A and eIF4E levels in responding and non-responding patients A-008 and A-004. Representative micrographs used for quantifications in Table 3. eIF4E (green), UGT1A (red) and DAPI (blue) in sorted bone marrow blasts are shown. AML blasts isolated by FACS and stained as described in the Online Supplementary Appendix. Patient A-008 (A) achieved a 4-month stable disease (SD) and patient A-004 (B) a progressive disease (PD). BT: before treatment; CT: completed treatment cycle. Dapi provided as a nuclear marker. Scale bars: 10 µm.