CD44 loss of function sensitizes AML cells to the BCL-2 inhibitor venetoclax by decreasing CXCL12-driven survival cues.

Acute myeloid leukemia (AML) has a poor prognosis under the current standard of care. In recent years, venetoclax, a BCL-2 inhibitor, was approved to treat patients who are ineligible for intensive induction chemotherapy. However, complete remission rates with venetoclax-based therapies are hampered by minimal residual disease (MRD) in a proportion of patients, leading to relapse. MRD is a result of leukemic stem cells being retained in bone marrow protective environments; activation of the CXCL12-CXCR4 pathway was shown to be relevant to this process. An important role is also played by cell adhesion molecules such as CD44, which has been shown to be crucial for the development of AML. Here we show that CD44 is involved in CXCL12 promotion of resistance to venetoclax-induced apoptosis in human AML cell lines and AML patient samples, which could be abrogated by CD44 knock down, knockout, or blocking with an anti-CD44 antibody. Split-Venus bimolecular fluorescence complementation showed that CD44 and CXCR4 physically associate at the cell membrane upon CXCL12 induction. In the venetoclax-resistant OCI-AML3 cell line, CXCL12 promoted an increase in the proportion of cells expressing high levels of embryonic stem cell core transcription factors (ESC-TFs: Sox2, Oct4, Nanog) abrogated by CD44 knockdown. This ESC-TF-expressing subpopulation which could be selected by venetoclax treatment, exhibited a basally enhanced resistance to apoptosis and expressed higher levels of CD44. Finally, we developed a novel AML xenograft model in zebrafish, which showed that CD44 knockout sensitizes OCI-AML3 cells to venetoclax treatment in vivo. Our study shows that CD44 is a potential molecular target for sensitizing AML cells to venetoclax-based therapies.

Improvement of Workflows and Assay Reproducibility by The Introduction of "Assay-Ready" Culturing of MDCK Cells for Transport Studies.

OBJECTIVE: In previous studies, we established and validated three Madin Darby Canine Kidney MDCKII cell lines, recombinantly modified with zinc finger nuclease (ZFN) technology. Here, we investigated the applicability of seeding these three canine P-gp deficient MDCK_ZFN cell lines, directly from frozen cryopreserved stocks without previous cultivation for efflux transporter and permeability studies. This technique is referred to as "assay-ready" and allows for highly standardized conduction of cell-based assays and shorter cultivation cycles. METHODS: To obtain a rapid fitness of the cells for that purpose, a very gentle freezing and thawing protocol was applied. Assay-ready MDCK_ZFN cells were tested in bi-directional transport studies and compared to their traditionally cultured counterparts. Long-term performance robustness, human effective intestinal permeability (Peff) predictability and batch to batch variability were assessed. RESULTS: Efflux ratios (ER) and apparent permeability (Papp) results were highly comparable between assay-ready and standard cultured cell lines with R2 values of 0.96 or higher. Papp to Peff correlations obtained from passive permeability with non-transfected cells were comparable independent of the cultivation regime. Long-term evaluation revealed robust performance of assay-ready cells and reduced data variability of reference compounds in 75% of cases compared to standard cultured MDCK_ZFN cells. CONCLUSION: Assay-ready methodology for handling MDCK_ZFN cells allows more flexibility in assay planning and reduces performance fluctuations in assays caused by cell aging. Therefore, the assay-ready principle has proven superior over conventional cultivation for MDCK_ZFN cells and is considered as a key technology to optimize processes with other cellular systems.

Conquering the beyond Rule of Five Space with an Optimized High-Throughput Caco-2 Assay to Close Gaps in Absorption Prediction.

Current drug development tends towards complex chemical molecules, referred to as "beyond rule of five" (bRo5) compounds, which often exhibit challenging physicochemical properties. Measuring Caco-2 permeability of those compounds is difficult due to technical limitations, including poor recovery and detection sensitivity. We implemented a novel assay, with optimized incubation and analytics, to measure permeability close to equilibrium. In this setup an appropriate characterization of permeability for bRo5 compounds is achievable. This equilibrated Caco-2 assay was verified with respect to data validity, compound recovery, and in vitro to in vivo correlation for human absorption. Compared to a standard assay, it demonstrated comparable performance in predicting the human fraction absorbed (fa) for reference compounds. The equilibrated assay also successfully characterized the permeability of more than 90% of the compounds analyzed, the majority of which were bRo5 (68%). These compounds could not be measured using the standard assay. Permeability and efflux ratio (ER) were highly predictive for in vivo absorption for a large set of internal bRo5 compounds. Reference cut-offs enabled the correct classification of high, moderate, and low absorption. This optimized equilibrated Caco-2 assay closes the gap for a high-throughput cellular permeability method in the bRo5 chemical space.

Mucin-Protected Caco-2 Assay to Study Drug Permeation in the Presence of Complex Biorelevant Media.

The poor solubility and permeability of compounds beyond Lipinski's Rule of Five (bRo5) are major challenges for cell-based permeability assays. Due to their incompatibility with gastrointestinal components in biorelevant media, the exploration of important questions addressing food effects is limited. Thus, we established a robust mucin-protected Caco-2 assay to allow the assessment of drug permeation in complex biorelevant media. To do that, the assay conditions were first optimized with dependence of the concentration of porcine mucin added to the cells. Mucin-specific effects on drug permeability were evaluated by analyzing cell permeability values for 15 reference drugs (BCS class I-IV). Secondly, a sigmoidal relationship between mucin-dependent permeability and fraction absorbed in human (fa) was established. A case study with venetoclax (BCS class IV) was performed to investigate the impact of medium complexity and the prandial state on drug permeation. Luminal fluids obtained from the tiny-TIM system showed a higher solubilization capacity for venetoclax, and a better read-out for the drug permeability, as compared to FaSSIF or FeSSIF media. In conclusion, the mucin-protected Caco-2 assay combined with biorelevant media improves the mechanistic understanding of drug permeation and addresses complex biopharmaceutical questions, such as food effects on oral drug absorption.