-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

662 Expression of Sf3b1-K700E accelerates the Development of Chronic Lymphocytic Leukemia in a Del(13q) Murine Model

Program: Oral and Poster Abstracts
Type: Oral
Session: 641. CLL: Biology and Pathophysiology, excluding Therapy: Genetic Models and Genomic Landscape of CLL and Richter Transformation
Hematology Disease Topics & Pathways:
Leukemia, Diseases, CLL, Lymphoid Malignancies
Monday, December 7, 2020: 12:15 PM

Bo Zhang, MD, PhD1, Prajish Iyer, PhD2*, Meiling Jin, PhD3*, Elisa Ten Hacken, PhD4, Zachary Cartun, BS4*, Kevyn Hart, BS3*, Laura Z. Rassenti, PhD5, Thomas J. Kipps, MD, PhD6, Donna S. Neuberg, ScD7, Ruben D. Carrasco, MD, PhD8*, Catherine J. Wu, MD9 and Lili Wang, MD, PhD3

1Department of Systems Biology, NCI Comprehensive Cancer Center, Beckman Research Institute | City of Hope, 1218 South 5th Avenue, Monrovia, CA, 91016, Monrovia, CA
2Systems Biology Department, City of Hope National Medical Centre, Monrovia, CA
3Department of Systems Biology, Beckman Research Institute, City of Hope, Monrovia, CA
4Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA
5Moores Cancer Center, University of California, San Diego, La Jolla, CA
6UCSD Moores Cancer Center, San Diego, CA
7Data Sciences Institute, Dana Farber, Boston
8Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA
9Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA

RNA splicing factor SF3B1 is one of the most recurrently mutated genes in chronic lymphocytic leukemia (CLL), but expression of this mutation alone in murine B cells does not result in CLL. This gene mutation is often subclonal and associated with poor survival. How this mutation impacts CLL progression remains elusive. Since SF3B1 mutation frequently co-occurs with chromosome 13q deletion (del(13q)), and mice with deletion of the Minimal Deleted Region (MDR) of del(13q) develop indolent CLL, we therefore asked whether co-expression of Sf3b1 mutation can accelerate the onset of CLL in this murine model. If so, how does Sf3b1 mutation mechanistically contribute to CLL.

To this end, we first crossed mice carrying conditional knock in allele Sf3b1-K700E and mice with conditional knockout of MDR. We then bred the offspring with CD19-Cre mice to generate cohorts of mice which have B cell-specific homozygous deletion of MDR with (DM) or without (MDR-MT) heterozygous Sf3b1-K700E. We monitored the onset of CLL by tracking of circulating B220+CD5+ CLL-like cells from peripheral blood with flow cytometry, starting at the age of 6-months and ending by 24-months. We detected CLL-like disease in 24% (6 of 25) of DM and 7.4% (2 of 27) of MDR-MT mice with disease presence in the spleen, bone marrow and lymph node, confirmed by flow cytometry and immunohistochemistry. The increased frequency of CLL in DM mice indicated that Sf3b1-K700E could accelerate CLL (Pearson Chi-Square 2-sided, p=0.098).

To elucidate how Sf3b1 mutation contributes to increased CLL penetrance, we performed integrated RNA sequencing (RNA-seq) and TMT proteomics analysis with splenic B cells derived from DM mice with and without CLL. We found that genes involved in MYC, cell cycle checkpoints and mTORC1 pathways are upregulated and enriched at both the RNA and protein levels when we compared DM-CLL cells to their DM B cell counterparts, indicating these cellular processes are involved in the onset of CLL. To further define the role of Sf3b1-K700E mediated alternative splicing in the activation of these pathways, we first identified candidate splicing isoforms (nfatc1, braf, depdc5, tsc2) through computational analysis of RNA-seq data and then validated the isoforms in an independent cohort of samples (n=3,). Functional annotation of how exactly these isoforms impact CLL is ongoing. Importantly, we also observed gene upregulation of mTORC1 pathway in human CLL cells with SF3B1 mutation and del(13q) when compared with normal B cells.

We next asked whether DM CLL cells are sensitive to inhibition of mTORC1 pathway and RNA splicing inhibition in vitro. We exposed DM B and CLL cells to either Temsirolimus (Tem, mTORC1 inhibitor), or H3B8800 (H3B, SF3B1 inhibitor) alone or in combination for 24 hours and then measured the cell viability with CellTiter-Glo assay. When compared to DMSO control, both Tem and H3B single treatments significantly inhibited the survival of DM CLL cells, but not DM B cells (all groups vs control, unpaired t test, p<0.01). Furthermore, an additive effect was observed in DM CLL cells when 1nM of H3B was combined with Tem treatment (IC50: 1.2nM vs. 135.2uM, unpaired t test p<0.001). We then tested the effects of both drugs in vivo using NSG mice engrafted with DM CLL cells. Mice treated with combination of Tem (15mg/kg, i.p, 5 days) and H3B (4mg/kg, gavage, 5 days) had a lower CLL burden in peripheral blood in comparison to either the single treatment or no drug treatment group (all groups vs. comb, p≤0.001). Furthermore, the combination treatment increased the survival of NSG mice engrafted with CLL cells compared to control (median survival: control vs. comb 15 vs. 34 days, log rank p<0.001). Importantly, when we exposed human CLL cells with both del(13q) and sf3b1 mutation (DM-CLL, n=3), or with del(13q) alone (n=2), or normal B cells (n=4) to the combination treatment in vitro, DM-CLL cells showed the highest sensitivity to the treatment (DM-CLL vs. all groups, p<0.05), suggesting that SF3B1 mutation may accelerate CLL with del(13q) through modulating RNA splicing and mTORC1 pathway.

Our study demonstrates that expression of Sf3b1-K700E could accelerate the development of CLL based on MDR deleted murine model through alternative RNA splicing and mTORC1 activation. This finding supports the use of an mTORC1 inhibitor together with RNA splicing inhibitor in the subset of CLL patients with both SF3B1 mutation and del(13q).

Disclosures: Kipps: Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding. Neuberg: Celgene: Research Funding; Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding. Wu: BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

*signifies non-member of ASH