Dose-Response Titration In a Bone Marrow Niche Model Reveals AKT As a Target That Modulates Sensitivity Of Myeloid Leukemia To Extracellular Matrix Mechanics: Implications In Drug Resistance
Authors: Shin Jae-Won , Mooney David J. 
Published: October 2019

Abstract

Background
Acute Myeloid Leukemia (AML) is a cell-autonomous disorder with genetic heterogeneity, and response to existing therapies is not curative. Emerging evidence pinpoints the bone marrow (BM) milieu as a non-autonomous contributor to the pathophysiology of myeloid leukemia, suggesting a novel therapeutic strategy to target the disease. While soluble and adhesion molecules from the BM regulating leukemia are beginning to be characterized, cells can also generate force and sense the mechanical properties of the BM niche. The BM niche is disrupted in some cases of leukemia, including the dysregulation of osteoblastic lineage cells that could lead to altered extracellular matrix mechanics and drug resistance. Deregulation of the serine/threonine kinase AKT has been associated with a number of cancers, including AML. Interestingly, AKT is required for sensing of matrix stiffness in some solid tumors, but its significance in leukemia is unknown. This study is based on the hypothesis that myeloid leukemic cell proliferation is regulated by matrix mechanics via AKT signaling pathway. We have developed a new model system to test this hypothesis, and to characterize drugs that specifically target the ability of leukemic cells to sense the physical properties of the BM niche.

Methods
The BM niche consists of heterogeneous micro-mechanics ranging from viscous to elastic regions. To model this in vitro, we used an alginate-based hydrogel conjugated with an integrin-binding RGD peptide to culture cells in a three-dimensional space, multi-well setting. Mechanical properties of the hydrogel were controlled by Ca2+ cross-linking and characterized by a rheometer. Human myeloid leukemia cell lines were characterized in this culture system: AML with MLL-AF9 translocations (MOLM-14 and NOMO-1), AML without MLL-AF9 (U-937), and Acute Promyelocytic Leukemia (HL-60). After encapsulating cells across different matrix mechanics, dose response analyses were performed.

Results
Without gel cross-linking, the alginate fluid recapitulates the known viscosity value of BM (40∼400 cP). Upon Ca2+ cross-linking, a viscoelastic solid is formed with a minimum elastic modulus at 50 Pa. All of the tested leukemic cells show 2∼3 fold increased cell proliferation in the soft solid (50 Pa) compared to the viscous fluid after one week culture. In contrast, increasing the stiffness of the solid further to 1000 Pa decreases proliferation by 1.5∼2 fold, suggesting a biphasic mechanical response from fluid to solid. Higher ligand density suppresses the cell number of HL-60 and U-937, while it increases that of MLL-AF9+ cells by ∼2 fold. On the other hand, primary normal CD34+BM cells are not sensitive to the transition from fluid to soft solid. The dose response curves of a DNA-synthesis inhibitor cytosine arabinoside show no change in potency or cooperativity across different matrix mechanics, indicating that higher doses of this drug are required to overcome increased leukemic cell number in soft solid relative to fluid. In contrast, pharmacological modeling predicts that the inhibition of bona-fide mechano-regulatory factors should eliminate the initial difference in cell number caused by matrix mechanics as the drug dose starts to increase. As a result, dose-response curves from different matrix mechanics will converge at a specific dose, followed by merging of the curves with higher doses. Dose response titration of MK-2206, a potent allosteric inhibitor of AKT phosphorylation at Ser473 (pS473-AKT), which was recently used in a Phase I study of solid tumors, indicates that AKT may fit the profile of mechano-regulatory targets in myeloid leukemia. MK-2206 decreases the cell number in the soft solid but not in the fluid until the dose reaches a point of convergence for the curves. As a result, the drug potency is increased by 2 fold from fluid to soft solid for both U-937 and MOLM-14, whereas the cooperativity is decreased by 3 fold for U-937. A similar trend was observed at the molecular level based on the dose-response curves of pS473-AKT expression evaluated by intracellular flow cytometry analyses.

Conclusions
The results suggest that AKT is a potential target to modulate sensitivity of myeloid leukemia to matrix mechanics. More broadly, this study provides a strategy to screen for drugs that target sensing of extracellular matrix mechanics by myeloid leukemia and overcome microenvironment-mediated drug resistance.

Disclosures:
No relevant conflicts of interest to declare.