Session: 506. Bone Marrow Microenvironment: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Acute Myeloid Malignancies, AML, Artificial intelligence (AI), Assays, Hematopoiesis, Diseases, Immune mechanism, Treatment Considerations, Immunology, Myeloid Malignancies, Biological Processes, Technology and Procedures, Molecular biology, Multi-systemic interactions, Pathogenesis, Imaging, Machine learning, Omics technologies
Methods: We cocultured AML cell lines (OCI-AML3 and OCI-AML2) with stromal cells (HS-27A) and inhibited several adhesion receptors (integrins α4, β2, αvβ3, and CD44) and downstream Rac signaling. Both, AML and stromal cells were subjected to transcriptome profiling, and scanning electron microscopy (SEM) to characterize topological properties. In addition, we used semi-automated microfluidic adhesion assays coupled to a machine-learning analysis algorithm. The algorithm classified cellular interactions with the substrate upon treatment with various stimuli and inhibitors at a single-cell level. Finally, we used the BEAT AML patient cohort (Bottomly et. al. Cancer Cell 2022) and set up a xenograft model treated with Venetoclax/Azacitidine (Ven/Aza) and an α-integrin α4 blocking antibody.
Results: In OCI-AML3 (NPM1mut DNMT3amut) cells, transcriptome profiling revealed a robust downregulation (log FC = - 0.87; P = 2.6 * 10-5) of SPARC, an extracellular matrix molecule and a counter ligand to VCAM-1 (Kelly et. al. Journal of Leukocyte Biology 2007) after inhibiting adhesion to stroma. Stroma contact induced a 21-fold upregulation in SPARC levels (P = 0.0009) in OCI-AML3, but not OCI-AML2 (NPM1wt DNMT3amut) cells. This expression was 8.8-fold downregulated (P = 0.007) upon integrin α4 inhibition. In addition, administering the Rac inhibitor, NSC23766, downregulated SPARC expression by 3.8-fold (P = 0.0032) indicating a Rac-dependent expression.
To address the function of SPARC, we established a microscopy-based adhesion assay using a machine-learning analysis pipeline and found that SPARC stimulation of OCI-AML3 cells enhanced adhesion to VCAM-1 by 1.7-fold (P = 0.015). This adhesion was abrogated by blocking integrin α4; indicating that SPARC stimulation affects integrin α4.
We investigated how this function manifested in cytoskeletal alterations on the AML-stroma surface using SEM. We observed that SPARC stimulation of OCI-AML3, but not OCI-AML2, led to the disassembly of cytoskeletal structures (termed ‘microvilli’) (P = 0.0013) in a Rac dependent manner (P = 0.048).
Finally, we stratified the BEAT AML cohort and filtered newly diagnosed NPM1mut DNMT3amut patients into quartiles. Patients with higher SPARC transcript levels had poor overall survival (median survival - 319 days; P = 0.0266) compared to patients with lower SPARC levels (median survival – undefined).
Ongoing in vivo studies with α-integrin α4 blocking antibody after Ven/Aza administration will determine if this sensitizes human NPM1-mutated cells expressing SPARC to Ven/Aza treatment.
Conclusion: In summary, using a comprehensive set of cell biological, transcriptomic and machine-learning tools, we propose a novel feedback mechanism whereby NPM1-mutated OCI-AML3 cells bind to the stroma leading to higher SPARC transcription. This transcriptional regulation is integrin α4 and Rac-dependent. Functionally, SPARC triggers cytoskeletal disassembly and enhances integrin α4/VCAM-1 mediated adhesion.
Disclosures: Shoumariyeh: Blueprint: Honoraria. Duque Afonso: Roche: Honoraria; Amgen: Honoraria; AstraZeneca: Honoraria; Beigene: Honoraria, Other: Travel support; Abbvie: Honoraria; Lilly: Honoraria; Janssen: Honoraria; Riemser: Honoraria; Ipsen: Honoraria; Sobi: Honoraria, Other: Travel support; Alexion: Other: Travel support; Gilead: Other: Travel support. Lübbert: Janssen: Research Funding; Cheplapharm: Research Funding; Otsuka: Honoraria; Syros Pharmaceuticals: Honoraria; AbbVie: Honoraria. Zeiser: Mallinkrodt: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Neovii: Consultancy; Novartis: Consultancy, Honoraria; Ironwood Pharmaceuticals, Inc.: Consultancy; Medac: Honoraria; Sanofi: Honoraria.