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1298 SPARC Stabilizes Integrin α4-VCAM-1 Interactions and Is Regulated By Intracellular Rac in NPM1-Mutated AML

Program: Oral and Poster Abstracts
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
Saturday, December 7, 2024, 5:30 PM-7:30 PM

Driti Ashok1,2*, Dennis Raith3,4*, Martin Helmstädter, PhD5,6*, Geoffroy Andrieux, PhD7*, Séverine Kayser5,6*, Sangya Chatterjee1*, Judith Treiber1*, Khalid Shoumariyeh, MD1*, Tim Struessmann, MD1*, Valentin Wenger1*, Julian Rapp, MD1*, Jesus Duque Afonso, MD1, Michael Lübbert, MD1, Robert Zeiser, MD1, Melanie Boerries, MD, PhD7,8*, Justus Duyster, MD1* and Tanja Nicole Hartmann, PhD1

1Department of Medicine I, Medical Center–University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
2University of Freiburg, Faculty of Biology, Freiburg, Germany
3LABMaiTE GmbH, Freiburg, Germany
4Department of Computer Science, University of Freiburg, Freiburg, Germany
5Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
6EMcore, Renal Division, Department of Medicine, University Hospital Freiburg, University Faculty of Medicine, Freiburg, Germany
7Institute of Medical Bioinformatics and Systems Medicine, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
8German Cancer Consortium (DKTK), Partner site Freiburg, a partnership between DKFZ and Medical Center - University of Freiburg, Freiburg, Germany

Introduction: Pathophysiological cues in AML can be mediated via an AML-stroma crosstalk leading to leukemic cell activation and retention. This interaction is shaped by soluble factors and direct cell-cell contact. Previously, we uncovered integrin α4β1 clusters expressed on AML cells as central adhesive hubs in this crosstalk, with VCAM-1 on the stromal side (Gutjahr et.al. Haematologica 2021). We have now dissected the signaling mechanism downstream of this adhesive hub, and have identified a Rac-dependent SPARC-α4β1 circuit in NPM1-mutated AML.

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.

*signifies non-member of ASH