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Background: Chronic myeloid leukemia (CML) pathogenesis is mainly driven by the oncogenic breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL) fusion protein. Since BCR-ABL displays abnormal constitutive tyrosine kinase activity, therapies using tyrosine kinase inhibitors (TKis) such as imatinib represent a major breakthrough for the outcome of CML patients. Nevertheless, the development of TKi resistance and the persistence of leukemia stem cells (LSCs) remain barriers to cure the disease, justifying the development of novel therapeutic approaches. Since the activity of histone deacetylase (HDAC) is deregulated in numerous cancers including CML, pan-HDAC inhibitors may represent promising therapeutic regimens for the treatment of CML cells in combination with TKi. Results: We assessed the anti-leukemic activity of a novel hydroxamate-based pan-HDAC inhibitor MAKV-8, which complied with the Lipinski's "rule of five," in various CML cells alone or in combination with imatinib. We validated the in vitro HDAC-inhibitory potential of MAKV-8 and demonstrated efficient binding to the ligand-binding pocket of HDAC isoenzymes. In cellulo, MAKV-8 significantly induced target protein acetylation, displayed cytostatic and cytotoxic properties, and triggered concomitant ER stress/protective autophagy leading to canonical caspase-dependent apoptosis. Considering the specific upregulation of selected HDACs in LSCs from CML patients, we investigated the differential toxicity of a co-treatment with MAKV-8 and imatinib in CML versus healthy cells. We also showed that beclin-1 knockdown prevented MAKV-8-imatinib combination-induced apoptosis. Moreover, MAKV-8 and imatinib cotreatment synergistically reduced BCR-ABL-related signaling pathways involved in CML cell growth and survival. Since our results showed that LSCs from CML patients overexpressed c-MYC, importantly MAKV-8-imatinib co-treatment reduced c-MYC levels and the LSC population. In vivo, tumor growth of xenografted K-562 cells in zebrafish was completely abrogated upon combined treatment with MAKV-8 and imatinib.

Aminoacyl-tRNA synthetases (ARSs) play essential roles in protein biosynthesis as well as in other cellular processes, often using evolutionarily acquired domains. For possible cooperativity and synergistic effects, nine ARSs assemble into the multi-tRNA synthetase complex (MSC) with three scaffold proteins: aminoacyl-tRNA synthetase complex-interacting multifunctional proteins 1, 2 and 3 (AIMP1, AIMP2 and AIMP3). X-ray crystallographic methods were implemented in order to determine the structure of a ternary subcomplex of the MSC comprising aspartyl-tRNA synthetase (DRS) and two glutathione S-transferase (GST) domains from AIMP2 and glutamyl-prolyl-tRNA synthetase (AIMP2GST and EPRSGST, respectively). While AIMP2GST and EPRSGST interact via conventional GST heterodimerization, DRS strongly interacts with AIMP2GST via hydrogen bonds between the α7–β9 loop of DRS and the β2–α2 loop of AIMP2GST, where Ser156 of AIMP2GST is essential for the assembly. Structural analyses of DRS–AIMP2GST–EPRSGST reveal its pivotal architecture in the MSC and provide valuable insights into the overall assembly and conditionally required disassembly of the MSC.

Organic anion transporting polypeptide 1B3 (OATP1B3) is a major influx transporter mediating the hepatic uptake of various endogenous substrates as well as clinically important drugs such as statins and anticancer drugs. However, molecular mechanisms controlling the membrane trafficking of OATP1B3 have been largely unknown. Several reports recently indicated the presence of a distinct, cancer-type OATP1B3 variant lacking the N-terminal 28 amino acids compared to OATP1B3 expressed in non-malignant hepatocytes. Interestingly, the cancer-type OATP1B3 variant is located predominantly in the cytoplasm, implicating the involvement of the N-terminal region of OATP1B3 in its membrane trafficking. In the current study, we set out to experimentally validate the importance of the N-terminal region of OATP1B3 and to identify responsible sequence motif(s) in that region. A number of truncation or point mutants of OATP1B3 were transiently expressed in HEK293T, HCT-8 or MDCK II cells and their expression in cytoplasmic and surface membrane fractions were analyzed by immunoblotting. Our results indicated that the N-terminal sequence of OATP1B3, in particular, at the amino acid positions between 12 and 28, may be indispensable in its membrane trafficking. Moreover, our results using a fusion construct indicated that the first 50 amino acids of OATP1B3 are sufficient for its membrane localization. The importance of the N-terminal region in membranous localization was shared among the other OATP1B subfamily members, OATP1B1 and rat Oatp1b2. Our efforts to identify the responsible amino acid(s) or structure motif(s) in the N-terminal region did not pinpoint individual amino acids or motifs with putative secondary structures. Our current findings however demonstrate that the N-terminal region is important for the membrane localization of the OATP1B subfamily members and should facilitate future investigations of the mechanisms involved in the regulation and membrane trafficking of these important transporter proteins.

Histone deacetylases (HDAC) are currently a group of validated targets for anticancer drug discovery and development. In our research program to find novel small molecules targeting these enzymes, we designed and synthesized two series of 3-hydroxyimino-2-oxoindoline- and 3- methoxyimino-2-oxoindoline-based N-hydroxypropenamides (3a-g, 6a-g). The results show that these propenamides potently inhibited HDAC2 with IC50 values in sub-micromolar range, approximately 10-fold lower than that of SAHA (also known as suberoylanilohydroxamic acid). Evaluation of cytotoxicity of these compounds in three human cancer cell lines revealed that most of the synthesized compounds were up to 5-fold more cytotoxic than SAHA. Docking studies showed that the compounds bound to HDAC2 at the binding site with higher binding affinities compared to SAHA. Our present results demonstrate that these novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides are potential for further development as anticancer agents.