Proteasome inhibitors (PIs) are valuable drugs for the treatment of multiple myeloma. Furthermore, PIs showed encouraging activity against other cancer types and are recently being involved in several clinical trials, also in the treatment of relapsed/refractory ALL and MLL 1, 2. However, they are not able to provide cures as monotherapy. Based on preclinical studies, the combination of PIs with histone deacetylase inhibitors (HDACi) are producing promising synergistic cytotoxicity. Aggresomes are used by many malignant cells as an alternative route to degrade proteins that accumulate after proteasome inhibition. However, this mechanism depends on HDAC6 to transport ubiquitinated proteins by microtubules. Thus, inhibition of the proteasome and HDAC6 results in synergistic anticancer activity by induction of apoptosis in cancer cells. The approach "one drug multiple targets" is gaining major consideration in drug discovery and has been termed polypharmacology. Despite the highly significant therapeutic relevance of combination therapies, potential advantages of a targeted therapy based on a single drug that acts through two independent modes of action include (a) a more predictable pharmacokinetic profile, (b) increased patient compliance, and (c) the simultaneous presence of the molecule in tissues where the active principle is needed to work. We therefore aimed at the design, synthesis and biological evaluation of proteasome-HDAC hybrid inhibitors.

The first-in-class dual proteasome-HDAC hybrid inhibitor 'RTS-V' was designed by hybridizing the pharmacophores of known HDAC6 selective HDACi 3 and the non-covalent proteasome inhibitor compound 16 4. Autodock 4.2 was used to predict the binding mode of RTS-V into the β5/β6 active site of 20S yeast proteasome (PDB ID: 3MG8) and HDAC6 (PDB ID: 5EDU).

The hybrid compound RTS-V was found to fit smoothly into the active site of the the β5 sub-unit of the yeast 20S proteasome and HDAC6 according to docking studies. RTS-V revealed submicromolar and preferential inhibition of HDAC6 in HDAC activity assays. The preferential inhibition of HDAC6 was further confirmed by WB analysis in accordance to Ricolinostat (an orally bioavailable HDAC6 inhibitor). Furthermore, we found that RTS-V specifically blocks chymotrypsin-like proteasome activity, in agreement to Bortezomib (BTZ). Next we screened RTS-V against a panel of more than 20 cell lines and determined the average IC50 values. The screen also included imatinib sensitive/resistant cell lines and three relapse primary BCP-ALL patient samples. Besides that, in three leukemic cells lines we could show that RTS-V inhibits cell proliferation, induces apoptosis, accumulates cells in S phase, induces early differentiation and inhibits colony formation. Like BTZ, RTS-V also acts through modulation of BCL-2, AKT, JNK and MAPK signaling pathways. PBMNCs and MSCs derived from five healthy individuals were treated with RTS-V and later determined the average IC50 values, which was found >20-fold higher than in leukemia cells. These results hint that the cytotoxic effect of RTS-V is significantly less pronounced on healthy cells as compared to leukemic cells.

In summary, we have developed a first-in-class proteasome-HDAC6 hybrid inhibitor (RTS-V) with activity against leukemic cell lines and primary relapse human BCP-ALL patient samples. Our next aim would be test its efficacy in in vivo studies. Taken together, RTS-V represents a promising starting point for future efforts towards the development of highly effective dual proteasome-HDAC6 inhibitors to overcome drug resistance, especially for the treatment of relapsed/refractory ALL.

  1. ClinicalTrials.gov Identifier: NCT02419755, NCT0131281 & NCT01075425

  2. Messinger, Y., Gaynon, P., Sposto, et al . (2012). Blood 120: 285-290.

  3. Wagner, F. F.; Olson, D. E.; Gale, et. al. J. Med. Chem. 2013, 56 , 1772-1776.

  4. Blackburn, C.; Gigstad, K, et. al. Biochem. J. 2010, 430 , 461-476.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.