Mebendazole Mediates Its Anti-Leukemic Effects By Proteasomal Degradation of GLI Transcription Factors Via Inhibition of HSP70/90-Chaperone Activity in Acute Myeloid Leukemia in a Preclinical and Clinical Setting

Aberrant activation of hedgehog signaling (HH) is associated with a wide variety of neoplasms, including acute myeloid leukemia (AML). The GLI transcription factors are the main downstream effectors of the HH signaling cascade and play a fundamental role in cancer development, progression and maintenance of leukemic stem cells, which are responsible for therapy failure and tumor relapse due to their resistance to chemotherapy. Moreover, the GLI transcription factors represent central hubs in oncogenic signaling and thus are an attractive therapeutic target for anti-cancer therapy. However non-canonical GLI activation by multiple oncogenic signaling pathways limit the use of SMO inhibitors, while treatment strategies directly targeting the GLI transcription factors are limited. Mebendazole (MBZ) is a commonly used anthelmintic drug with a favorable toxicity profile. MBZ has been shown to exhibit strong anti-tumor effects in different cancer entities, including AML.

We treated AML cell lines and primary AML samples with different MBZ concentrations and could show a dose-dependent effect on the proliferation, colony formation and apoptosis in AML cells. In addition, therapeutic synergy between cytarabine and MBZ was demonstrated in OCI-AML3 cells with a combination index <1 and a dose reduction index (DRI) of 2.5- to 23.0-fold. GLI1 and -2 protein levels were clearly reduced in AML cell lines 24h after MBZ exposure, whereas mRNA levels did not decrease. To study the functional activity of GLI transcription factors, we transduced various AML cell lines with a construct where luciferase expression is under the control of the GLI promoter. MBZ strongly inhibited GLI signaling activity in a dose-dependent manner. Furthermore, inhibiting the 26s proteasome with bortezomib partly abolished the effect of MBZ on GLI1 and -2 protein levels and GLI activity, indicating that MBZ promotes their proteasomal degradation. The HSP70/HSP90-based chaperone machinery stabilizes a spectrum of client substrates for protein folding, including transcription factors. Inhibition of either HSP90 or HSP70 with small-molecule inhibitors VER-155008 and PU-H71 resulted in markedly reduction of GLI1 and -2 protein levels in Western Blots. Next, we investigated the impact of MBZ on HSP70/HSP90-mediated refolding of the heat-denatured firefly luciferase. In cell-lines constitutively expressing a firefly luciferase, the luciferase signal was recovered without inhibitor following a heat-shock, but incubation of AML cells with MBZ or specific HSP inhibitors impaired recovery of the signal, suggesting strong inhibition of HSP70/90-mediated luciferase refolding. This indicates that the anti-leukemic activity of MBZ is at least partly mediated through GLI protein destabilization by inhibition of cooperative HSP70/HSP90-chaperone activity. This is further supported by our observation that MBZ activates HSF-1, a major regulator of the heat shock pathway. MBZ may address additional pathways by HSP70/HSP90 inhibition such as FLT3-ITD, MYB or MYC degradation and inactivation of ERK and AKT as shown by us and others.

To further evaluate if MBZ is a suitable GLI inhibitor in clinical practice, we transferred these findings into the clinical setting by treating two patients with refractory AML with MBZ monotherapy in an off-label setting. We demonstrated an effective MBZ concentration in the plasma using a modified plasma inhibitory assay (PIA) by incubating an indicator cell lines carrying the GLI luciferase promoter transgene with the patient's plasma. In one patient, a clear continuous decrease in leukemic blasts in peripheral blood was noted. In this patient a reduction in the luciferase activity in the PIA assay and a fast reduction in GLI2 levels in peripheral leukemic blood were detected. Moreover, a healthy volunteer ingested MBZ at a dose of 50 mg/kg divided in two doses at time 0h and 12h. Blood was drawn at 4h and at 24h. PIA results indicated a biological active plasma concentration.

Taken together, our results demonstrate that MBZ functions as an effective GLI inhibitor with strong anti-leukemic activity in a clinical setting and encourage for further studies to translate the scientific findings into clinical practice. A clinical study of mebendazole plus low dose cytarabine in patients with refractory AML is planned.

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