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1006 Mice Expressing MYC and NrasQ61R in Germinal Center B Cells Develop Highly Aggressive Multiple Myeloma

Program: Oral and Poster Abstracts
Type: Oral
Session: 652. Myeloma: Pathophysiology and Pre-Clinical Studies, excluding Therapy: Molecular Mechanisms of Myelomagenesis and Its Dependencies
Hematology Disease Topics & Pathways:
Diseases, Animal models, Bone Marrow Failure, Biological Processes, Study Population, Clinically relevant, pathogenesis
Monday, December 3, 2018: 7:00 PM
Ballroom 20A (San Diego Convention Center)

Adhithi Rajagopalan, BS1,2, Zhi Wen, PhD1*, Quinlan Furumo3*, Erik A. Ranheim, MD, PhD4, Remington Finn1,5*, Evan Flietner, BS6,7*, Grant Yun1,5*, Yun Zhou, MS1*, Mei Yu, PhD3*, Natalie Scott Callander, MD6, Marta Chesi, PhD8, P. Leif Bergsagel, MD8, Robert Burns, Ph.D.3*, Demin Wang, PhD 3,9, Fotios Asimakopoulos, PhD, MBBChir,6 and Jing Zhang, PhD1

1McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI
2Cellular and Molecular Biology Program, University of Wisconsin-Madison, Madison, WI
3Blood Research Institute, BloodCenter of Wisconsin, part of Versiti, Milwaukee, WI
4Department of Pathology & Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, University of Wisconsin Carbone Cancer Center, Madison, WI
5Department of Biochemistry, University of Wisconsin-Madison, Madison, WI
6Division of Hematology/Oncology, Department of Medicine, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI
7Cellular and Molecular Pathology Program, University of Wisconsin-Madison, Madison, WI
8Department of Medicine, Mayo Clinic Arizona, Scottsdale, AZ
9Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,770 in 2018, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency, suggesting that additional genetic mutations are required for the malignant MM progression.

Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre or NrasLSL Q61R/+; IgG1-Cre. To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly aggressive MM, which was characterized by high proliferative index, hyperactivation of AKT and ERK pathways, and disease hallmarks (e.g. osteolytic lesions, anemia, and kidney injury). VQ myeloma was readily transplantable into serial syngeneic recipients. In our preliminary study, combined Bortezomib and AZD6244 treatment attenuated MM phenotypes and prolonged the survival of VQ recipients.

To facilitate easy molecular or genetic manipulations of VQ MM cells, we established two cell lines from primary cells. These cells could be easily infected (see another abstract from our co-author, Dr. Asimakopoulos’s group). The cultured MM cells also express high levels of CD155, a ligand of immune checkpoint TIGIT, which has been recently reported to mediate a significant immune checkpoint blockade in MM patients.

We further investigated the transcriptional signature of VQ MM cells (from bone marrow [BM] and from lymph nodes [LN]) vs wild-type (WT) plasma cells using the Illumina Bio-Rad’s Single-Cell Sequencing platform. Illumina’s Sure Cell app was used to de-multiplex samples, process barcodes and perform single-cell 3’ gene counting. The processed data were then analyzed using Seurat R package to yield cluster information and their associated enriched genes. Our results revealed distinct clusters of CD138+ MM and WT cells, and also showed high similarity & overlap between BM & LN MM samples. Consistent with the genetic changes introduced to VQ MM cells, both Myc and Nras expression levels were significantly elevated in MM clusters compared to WT clusters. We also found that transcriptional levels of several potential therapeutic targets were significantly elevated in MM clusters, including Integrin alpha 4 and metabolic enzymes. Currently we are validating the scRNA-Seq results.

Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. The VQ mouse model represents an important innovation that will serve as a platform to investigate pathogenesis of resistant and refractory multiple myeloma and allow for testing the efficacy of novel therapeutic agents.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH