E417: Identifying druggable targets in desmoid fibromatosis using an shRNA screen

ePoster Abstract Author(s)

Katherine Prendergast, MD (she/her/hers)

Surgical Research Fellow
Kristen Ann Carr Sarcoma Biology Laboratory, Department of Surgery, Memorial Sloan Kettering Cancer Center
New York, New York, United States

Submitter(s)

Katherine Prendergast, MD (she/her/hers)

Surgical Research Fellow
Kristen Ann Carr Sarcoma Biology Laboratory, Department of Surgery, Memorial Sloan Kettering Cancer Center
New York, New York, United States

Author(s)

Katherine Prendergast, MD (she/her/hers)

Surgical Research Fellow
Kristen Ann Carr Sarcoma Biology Laboratory, Department of Surgery, Memorial Sloan Kettering Cancer Center
New York, New York, United States
JH

Jia Hu, PhD

Senior Research Scientist
Kristen Ann Carr Sarcoma Biology Laboratory, Department of Surgery, Memorial Sloan Kettering Cancer Center, United States
VT

Vladislav Tsiperson, PhD

Research Scientist
Kristen Ann Carr Sarcoma Biology Laboratory, Department of Surgery, Memorial Sloan Kettering Cancer Center, United States
SK

Sara Kryeziu, MD

Surgical Research Fellow
Kristen Ann Carr Sarcoma Biology Laboratory, Department of Surgery, Memorial Sloan Kettering Cancer Center, United States
MR

Marco Russo, PhD

Scientific Manager
Gene Editing and Screening Core, Memorial Sloan Kettering Cancer Center, United States
RG

Ralph Garippa, PhD

Director
Gene Editing and Screening Core, Memorial Sloan Kettering Cancer Center, United States
MH

Meera Hameed, MD

Chief of Surgical Pathology, Memorial Sloan Kettering Cancer Center
Bone and Soft Tissue Pathology Service, Department of Pathology, Memorial Sloan Kettering Cancer Center; Department of Pathology, Weill Cornell Medical College, United States
SS

Samuel Singer, MD, FACS

Chief, Gastric and Mixed Tumor Service, Department of Surgery, Memorial Sloan Kettering
Kristen Ann Carr Sarcoma Biology Laboratory, Department of Surgery, Memorial Sloan Kettering Cancer Center; Department of Surgery, Weill Cornell Medical College, United States
AC

Aimee Crago, MD, PhD

Associate Attending Surgeon
Kristen Ann Carr Sarcoma Biology Laboratory, Department of Surgery, Memorial Sloan Kettering Cancer Center; Gastric and Mixed Tumor Service, Department of Surgery, Memorial Sloan Kettering Cancer Center; Department of Surgery, Weill Cornell Medical College, United States

Introduction: Desmoid-type fibromatosis (DF) is a rare disease associated with activating mutations affecting b-catenin. It does not metastasize but local progression can cause significant morbidity. This study sought to identify novel therapies for patients with aggressive forms of DF by performing a druggable genome screen.

Methods: DF cell lines were validated by Sanger sequencing and immortalized with TERT. A lentiviral shRNA druggable genome library was introduced into DF cells and construct enrichment assessed after 10 passages using edgeR. Gene expression, cell proliferation, cell cycle phase, protein levels and nuclear concentrations were assessed by RNA-Seq, CyQuant, Guava Cell Cycle assay, immunoblot and NE-PER reagents, respectively, after drug treatment or lentiviral-encoded shRNA delivery.

Results:
The screen identified a total of 75 enriched and 20 depleted genes (p< 0.05). Most enriched genes included TP53 and CDKN1A (p≤3.3x10^-9); depleted genes included ETF1, RTPL37, MDM2, and XPO1 (p≤0.01), which encodes exportin-1 and is reported to regulate b-catenin subcellular localization. Knockdown (KD) of XPO1 decreased proliferation 54-56% in DF cells (p<0.04) in validation experiments as did the exportin-1 inhibitor Selinexor (IC₅₀91nM [CI: 70-120nM] vs 270nM [CI:210-350nM] in mesenchymal stem cells). Neither XPO1 KD nor Selinexor treatment altered nuclear vs cytoplasmic localization of b-catenin, and RNA-Seq performed on XPO1 KDs showed only 3.5% of dysregulated genes were also observed in CTNNB1 KDs. Instead, gene set enrichment analysis suggested exportin-1 alters cell cycle progression. XPO1 KD or Selinexor treatment increased MDM2, p53, and p21 in DF cells, as well as the proportion of cells observed in G₁(60-72% vs 52% in KDs vs scramble control; 88% after treatment with 125nM Selinexor vs 48% in control cultures). Nuclear levels of both p53 and MDM2 were increased after Selinexor treatment.

Conclusions: An shRNA screen nominated druggable targets in DF, including XPO1. Exportin-1 knockdown and Selinexor treatment did not alter cellular localization of b-catenin but instead affected nuclear localization and total levels of MDM2 and p53 while inducing G1
cell cycle arrest. Exportin-1 regulates MDM2-p53 axis in DF cells and is a promising therapeutic target for DF patients. These results advocate testing Selinexor in a phase 2 clinical trial of progressing DF patients.

Learning Objectives:

Understand that, although desmoid fibromatosis does not metastasize, it can cause significant local morbidity.
Describe shRNA screening as a method for identifying targetable vulnerabilities in a population of cells.
Recognize exportin-1 as a regulator of the MDM2-p53 axis in desmoid fibromatosis and a promising therapeutic target for patients with desmoid fibromatosis.