Anti-Neoplastic Effect Of High Affinity and Covalent-Binding Microtubule Stabilizing Agents In AML Cells Sensitive and Resistant By ABC-Transporter Overexpression
Authors: Pera Benet , Jordi Michel , Hoya-Arias Ruben , Díaz José Fernando , Fang Weishuo , Altmann Karl-Heinz , Moore Malcolm A.S. 
Published: November 2019

Abstract
Poor outcomes in patients with acute myeloid leukemia (AML) are related to the high proportion of patients with chemorefractory disease or relapse after an initial response due to the development of resistance. This treatment failure is frequently due to the persistence of a cell population that is inherently resistant to classical chemotherapeutic agents through different mechanisms, in which an increased efflux of these agents via transmembrane proteins of the ATP-binding cassette (ABC) family is one of the best recognized. Classically, the main approach to overcome the transport effect has involved the co-administration of competitive inhibitors of these transporters. However, the large number of failed clinical trials involving these inhibitors has demonstrated the necessity to adopt different strategies.
We therefore hypothesized that molecules with covalent binding and/or high binding affinities well above their affinities for ABC-transporters will overcome the transport efflux, allowing AML cells to recover drug-sensitivity. To test this we have evaluated the anti-neoplastic activity of a series of fourteen synthetic paclitaxel analogues called chitaxes (CTXs), as well as the marine compound with tubulin-covalent binding ability zampanolide (ZMP) and seven novel synthetic derivatives. The CTXs compounds were designed combining the most favorable substitutions that increased the binding free energies up to 500-fold the paclitaxel binding affinity. For the ZMP derivatives, the overall rationale was to produce structurally simplified analogs that would be easier to synthesize and also be chemically more stable than ZMP.
First we determined the GI50,72h of the studied molecules in MV4-11 and HL-60 AML-cell lines using alamarBlue, resulting all of them active in the subnanomolar to low-micromolar range. ZMP and CTX number 40 (CTX-40) presented the highest activities with GI50,72h values of 0.16±0.03 nM in MV4-11 and 0.38±0.2 nM in HL-60, and 0.22±0.03 nM in MV4-11 and 0.27±0.04 nM in HL-60, respectively. With the exception of CTXs number 6, 30, 34 and 35, which shown GI50,72h values around 10 nM, the remaining paclitaxel derivatives shown values around 1 nM. On the other hand, the novel ZMP analogues presented activities from 30 nM to 2 μM. ZMP and CTX-40 were the more active compounds of the studied series, and they also shown activities around 8, 40 and 80 times higher than paclitaxel, daunorubicin and cytarabine, respectively. We then performed an early-apoptosis evaluation, treating the cells with ZMP or CTX-40 at their GI50,72h and staining with 7-AAD/Annexin-V, and we determined that more than 75 % of the cells entered apoptosis after 48 h of drug-incubation. Cell cycle analysis revealed that the addition of ZMP or CTX-40 induced a shift from G1 to G2/M as early as 12 h after treatment in a concentration dependent manner. In order to validate our working hypothesis, we evaluated the activity of ZMP and CTX-40 in resistant cells with P-glycoprotein overexpression, and we obtained low resistance indexes (GI50,72h resistant / GI50,72h sensitive) that revealed both compounds to be active in such resistant cells. To investigate the effect of these molecules on normal human hematopoietic progenitors and stem cells, we performed colony forming unit (CFU) assays and cobblestone area-forming (CAFC) assays, respectively. ZMP seems to be more active than CTX-40 in inhibiting progenitor differentiation as well as the growth of hematopoietic stem cells, however both compounds allowed the survival of these normal cells in the presence of drug concentrations up to 2 fold of their GI50,72h values. We are now evaluating the efficacy of these molecules on leukemic cells and their toxic effects on the healthy hematopoietic cells at the same time, by co-culturing HL-60 GFP-transduced cells with cord blood-CD34+ cells and distinguishing the leukemic colonies from the healthy ones under fluorescence microscopy. We are also exploring different combinations of ZMP or CTX-40 with daunorubicin or cytarabine, with the aim of identifying a combination that could allow reduction of the dose of the approved drug and increase the therapeutic window.
Our data presents ZMP and CTX-40 as candidates to be considered for therapeutic use in AML, not only due their promising in vitro activity but also because of their ability to retain such activity in resistant cell lines associated with P-glycoprotein overexpression.

Disclosures:
No relevant conflicts of interest to declare.