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4 Secreted Mutant Calreticulins As Rogue Cytokines Trigger Thrombopoietin Receptor Activation Specifically in CALR Mutated Cells: Perspectives for MPN Therapy

Program: General Sessions
Session: Plenary Scientific Session
Hematology Disease Topics & Pathways:
Diseases, Biological Processes, MPN, thrombocythemia, Myeloid Malignancies, hematopoiesis, molecular interactions, pathogenesis, signal transduction
Sunday, December 2, 2018, 2:00 PM-4:00 PM
Hall AB (San Diego Convention Center)

Christian Pecquet, PhD1*, Thomas Balligand, MD1*, Ilyas Chachoua, PhD1*, Anita Roy, PhD2*, Gaelle Vertenoeil, MD2*, Didier Colau, PhD1*, Emanuel Fertig, MD3*, Caroline Marty, PhD4*, Harini Nivarthi, PhD5*, Jean-Philippe Defour, BPharm, PhD6*, Erica Xu7*, Eva Hug, PhD8*, Heinz Gisslinger9*, Bettina Gisslinger9*, Martin Schalling, MSc9*, Ilaria Carola Casetti, MD10*, Elisa Rumi, MD10*, Daniela Pietra, Ph.D.11*, Chiara Cavalloni, MD11*, Luca Arcaini, MD12,13*, Mario Cazzola14, Norio Komatsu, MD, PhD15, Yoshihiko Kihara, PhD16*, Yoshitaka Sunami, MD, PhD16*, Yoko Edahiro, MD, PhD16*, Marito Araki, PhD17*, Isabelle Plo18*, William Vainchenker, MD, PhD4*, Robert Kralovics, PhD19 and Stefan N Constantinescu, MD1

1Ludwig Institute for Cancer Research, Brussels, Belgium
2Brussels, Ludwig Institute for Cancer Research, Brussels, Belgium
3National Institute of Pathology Victor BabeČ™, Bucharest, Romania
4Gustave Roussy, INSERM, UMR1170, Villejuif, France
5CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
6Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium
7MyeloPro Research and Diagnostics GmbH, Vienna, Austria
8MyeloPro Diagnostics and Research GmbH, Vienna, Austria
9Department of Internal Medicine I, Division of Hematology and Blood Coagulation, Medical University of Vienna, Vienna, Austria
10Department of Molecular Medicine, University of Pavia, Pavia, Italy
11Department of Hematology Oncology, IRCCS Policlinico San Matteo Foundation, Pavia, Italy
12Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
13Fondazione IRCCS Policlinico San Matteo and Department of Molecular Medicine, University of Pavia, Pavia, Italy
14University of Pavia, Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy
15Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan
16Department of Hematology, Juntendo University Graduate School of Medicine, Tokyo, Japan
17Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan
18INSERM, U1170, Gustave Roussy, Villejuif, France
19Department of Laboratory Medicine, Medical University of Vienna, Research Center For Molecular Medicine of the Austrian Academy of Sciences, VIENNA, Austria

Background
Mutant calreticulins carrying the sequence translated after a +1 frameshift at the C-terminus are major drivers of myeloproliferative neoplasms (MPNs). These mutant CALRs bind and activate TpoR/MPL in cells co-expressing TpoR and mutant CALRs, resulting in persistent JAK2-STAT5 signaling. Whether mutant CALR proteins are secreted, thus acting in trans on other cells, is not known.

Aims
Our objectives were to: 1) assess the direct TpoR-mutant CALR interaction both when expressed in the same or in different cells; 2) determine whether mutant CALRs are secreted; and 3) determine whether mutant CALR can act as extracellular cytokines.

Methods
Engineered CALR and TpoR mutants were analyzed by a combination of biochemical approaches (bioluminescence resonance energy transfer, recombinant protein production), functional assays (cell growth and transcriptional assays, flow cytometry, primary megakaryocytic clonogenic assay, analysis of CALR del52 knock-in mice) and cell imaging (confocal microscopy, flow cytometry and immuno-gold electron microscopy). Secreted CALRs were determined by ELISA using mutant specific antibodies.

Results
1) Two systems provided evidence that mutant CALRs and TpoR directly interact. First, using Nano-BRET in cells co-expressing N-terminally fused TpoR or EpoR with Nano-luciferase and mutant or WT CALR C-terminally tagged with HaloTag that is bound to the 618-ligand fluorophore, we show that TpoR and mutant CALRs interact in a complex whether the two proteins are within 10 nm. The interaction does not occur between TpoR and WT CALR, or between EpoR and mutant or WT CALRs. Second, expressing mutant CALR and TpoR extracellular domain in S2 Drosophila Schneider cells showed that stable complex formation requires immature high mannose structure on TpoR. Lastly, we could detect surface expression of the TpoR/CALRdel52 complex using proximity ligation assay with anti-TpoR and anti-mutant CALR antibodies in CRISPR/Cas9 engineered UT7/Tpo cells that express endogenous mutant CALR and TpoR levels.

2) We used flow cytometry, confocal immunofluorescence and immunogold electron microscopy and could show that mutant CALRs are trafficking via cis-, medial- and trans-Golgi to the cell-surface and are secreted, independently from TpoR expression. Importantly, mutant CALRs are also secreted in CALR mutated MPN patients as determined by mutant CALR-specific ELISA assay in patient plasma (mean plasma level 24.6 ng/ml, range 0-156.5 ng/ml). In the 113 evaluated CALR mutated patients from different centers the plasma mutant CALR levels correlated with CALR mutant allele burden (P<0.001). Secreted mutant CALR can also be found in plasma from knock-in CALR del52 mice.

3) We show that recombinant mutant CALR can act as a cytokine and specifically stimulate JAK2-STAT5 pathway in cells that carry the TpoR at the surface. Using Nano-BRET, we could demonstrate that extracellular mutant Halo-tagged CALR can specifically bind in trans to the cell-surface TpoR fused with Nano-luciferase, but not to EpoR fused with Nano-luciferase. This binding and the subsequent JAK2 activation were obtained at levels of around 100-150 ng/ml only in cells exposing at the cell-surface TpoR with at least one immature N-linked sugar. This can be accomplished by co-expressing in the reporter cells non-tagged mutant CALR, which will promote cell-surface localization of partially immature TpoR. The effect of exogenous mutant CALR could involve both stabilization of the endogenous cell-surface mutant CALR-TpoR complexes and binding to unoccupied immature TpoRs.

Conclusion
We show that mutant CALRs directly interact with TpoR and also are secreted and can act as rogue cytokines, leading to activation of cells carrying TpoR. Activation of TpoR in trans is efficient at mutant CALR levels similar to those detected in patients when target cells co-express heterozygous mutant CALR and TpoR, where endogenous mutant CALR transports to the surface TpoR with immature glycosylation. Thus, secreted mutant CALRs is predicted to expand the MPN clone. Given that cell-surface mutant CALR in TpoR expressing cells is crucial for oncogenicity, and that mutant CALRs are also secreted correlating with allele burden, we discuss how antibodies and other immunotherapy approaches could specifically target the mutant CALR MPN clone.

Disclosures: Xu: MyeloPro Research and Diagnostics GmBH: Employment. Hug: MyeloPro Diagnostics and Research GmbH: Employment. Gisslinger: Janssen: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Shire: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Kralovics: MyeloPro Diagnostics and Research GmbH: Equity Ownership. Constantinescu: MyeloPro Research and Diagnostics GmbH: Equity Ownership; Novartis: Consultancy; Novartis: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; Personal Genetics: Membership on an entity's Board of Directors or advisory committees; AlsaTECH: Equity Ownership.

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