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E-Selectin Ligand Expression increases with Progression of Myeloma and Induces Drug Resistance in a Murine Transplant Model, Which is Overcome By the Glycomimetic E-Selectin Antagonist, GMI-1271

Alessandro Natoni, Ph.D.1, Theodore A.G. Smith, Ph.D. 2, Niamh Keane, MB, MRCP1, Silvia Locatelli-Hoops, Ph.D.2, Isabela Olivera2, William E. Fogler, Ph.D. 2, John L. Magnani Ph.D. 2, Michael O’Dwyer, MD, FRCPath1.

1National University of Ireland Galway, Ireland, 2GlycoMimetics, Inc., Rockville, MD.

Objectives

• Myeloma is an incurable disease of plasma cells that spreads from one to multiple bone sites

• A key feature of MM is the intimate connection and dependency of malignant cells with the bone marrow (BM) microenvironment

• E-selectin regulates the Step-1 rolling interaction required for tissue-specific cell migration and is constitutively expressed in the specialized BM endothelium where it regulates the migration and recruitment of Human Stem/Progenitor cells (HSPCs) to the BM

• Functional E-Selectin ligands are characterised by reactivity with the HECA-452 antibody

• Assess the in vitro functionality of E-selectin ligands on Multiple Myeloma cells • Determine the tumorigenic potential of HECA-452 enriched Multiple Myeloma cells in

xenograft mouse models• Examine the expression of the HECA-452 epitope on Multiple Myeloma primary

samples

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MM Cell lines Enriched for HECA-452 interact with E-Selectin under static and dynamic conditions. This interaction can be

blocked by the small molecule inhibitor GMI-1271

Various populations of RPMI8226 cells with different percentage HECA-452 positive cellswere selected (A) . These RPMI8226 populations display adhesion (B) and rolling (C) on E-Selectin proportionally to the HECA-452 positive cells. (B) Bars represent mean±SD of one replicate. (C) Bars represent mean±SD of two independent repeats. Rolling on E-Selectin was examined in MM1SParenta and MM1SHECA-452 cells in presence of 10 µM E-Selectin inhibitor GMI-1271(E) . GMI-1271 completely blocked rolling on E-Selectin. Bars represent mean±SD of three independent repeats. E-Selectin Ab: 15 µg/ml, EDTA: 5 mM. Rolling assay was performed on E-Selectin coated microchannels at shear stress of 0.5 dyne/cm2. Static adhesion assay was performed in 96 well plates coated with 15 µg/ml crosslinked E-Selectin/IgG Chimera.

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MM Cell lines Enriched for HECA-452 give rise more aggressive disease and are resistant to Bortezomib in vivo

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Female SCID beige mice were injected i.v. with either MM1SParental or MM1SHECA-452 (5x105 cells, n=8/group) and followed for survival (A) . Mice transplanted with MM1SHECA-452 had more aggressive disease with significantly shorter survival compared to those transplanted with MM1SParental. In contrast to the parental cell line, mice engrafted with MM1SHECA-452 demonstrated a marked resistance to BTZ treatment that could be reverted by GMI-1271 (B).

Primary Myeloma cells are positive for HECA-452 with high percentage of positive cells in the Peripheral Blood which

increases with disease progression

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Bone Marrow (BM) and/or Peripheral Blood (PB) were obtained following informed consent from patients with MM and plasma cells (CD38+/CD138+) were analyzed for E-selectin ligand expression by flow cytometry using the HECA-452 antibody. A consistently higher proportion of circulating MM cells isolated from patient PB express HECA-452 when compared with paired BM samples (A,N=14), with a median difference of 33% (Wilcoxon signed rank test, p=0.02). (B) HECA-452 expression of MM in PB was significantly higher (on average 40% higher) in samples taken at relapse vs. diagnosis, (unpaired t test, p = 0.0008)

Multiple Myeloma and E-Selectin

Hypothesis and Methodology

To study the role of E-Selectin in Myeloma biology, we have selected Myeloma cells highly enriched for E-Selectin ligands using the HECA-452 antibody which marks the E-Selectin binding activity

Given the importance of the BM microenvironment in MM disease and the requirement of E-selectin for homing into the BM niche, the role of E-Selectin and their ligands for the biology of MM were examined

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Conclusions

• Multiple Myeloma cells express functional E-Selectin ligands• HECA-452 positivity tends to be higher in circulating Multiple

Myeloma cells of Relapse Patients• HECA-452 Enriched cells display aggressive disease and

resistance to bortezomib in vivo which can be reverted by GMI-1271

• E-selectin ligand bearing cells play an important role in dissemination, disease progression and drug resistance in Myeloma

Acknowledgments and Funding

We would like to acknowledge all members of Prof O’Dwyer’s laboratory, Prof Lokesh Joshi and Dr Michelle Kilcoyne from the Glycobiology laboratory and Dr Shirley Hanley from the Flow Cytometry Core Facility at the NUIGalway, Biosciences. We would like to thank the University Hospital Galway and in particular members of the Haematology/BTE staff and all the patients who have contributed to this research. This research is funded by the HRB CSA 201210 awarded to Prof O’Dwyer

Disclosures

Theodore A.G. Smith, Silvia Locatelli-Hoops, Isabela Olivera, William E. Fogler and John L. Magnani are employees of GlycoMimetic Inc., Michael O’Dwyer