-Author name in bold denotes the presenting author
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4135 First-Hit SETBP1 Mutations Cause a Myeloproliferative Disorder with Bone Marrow Fibrosis

Program: Oral and Poster Abstracts
Session: 602. Myeloid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research, MPN, Chronic Myeloid Malignancies, hematopoiesis, Diseases, Myeloid Malignancies, Biological Processes, Human
Monday, December 11, 2023, 6:00 PM-8:00 PM

Ilaria Crespiatico1*, Mattia Zaghi, PhD2*, Cristina Mastini, PhD3*, Deborah D'Aliberti, PhD4*, Mario Mauri, PhD3*, Carl Mirko Mercado, MSc3*, Diletta Fontana, PhD5*, Silvia Spinelli, MSc4*, Valentina Crippa, MSc3*, Elena Inzoli, MD6*, Beatrice Manghisi, MD4,6*, Ivan Civettini, MD7,8*, Daniele Ramazzotti, PhD4*, Valentina Fabiola Ilenia Lavoro, MD4,9*, Michele Gengotti, MD10*, Virginia Brambilla, MD11*, Andrea Aroldi, MD12*, Federica Banfi, MSc13*, Cristiana Barone, PhD14*, Roberto Orsenigo, MSc15*, Ludovica Riera, BSc, PhD16*, Mara Riminucci, MD17*, Alessandro Corsi, MD17*, Massimo Breccia18*, Alessandro Morotti, MD, PhD19, Daniela Cilloni, MD19*, Aldo Roccaro, MD, PhD20, Antonio Sacco, MSc21*, Fabio Stagno, MD, PhD22, Marta Serafini, PhD23*, Federica Mottadelli, MSc24*, Giovanni Cazzaniga, PhD25,26, Fabio Pagni, MD27*, Roberto Chiarle, MD16,28, Emanuele Azzoni, PhD14*, Alessandro Sessa2*, Carlo Gambacorti-Passerini, MD7,8, Elena Maria Elli, MD29*, Luca Mologni, PhD7* and Rocco Piazza, MD, PhD4,12*

1Department of Medicine and Surgery, University of Milano Bicocca, Monza, Italy
2Stem Cell and Neurogenesis Unit, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milano, ITA
3Department of Medicine and Surgery, University of Milano-Bicocca, Monza, ITA
4Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
5Department of Medicine and Surgery, Università degli Studi Milano-Bicocca, Monza, ITA
6Hematology Division and Bone Marrow Unit, Fondazione IRCCS San Gerardo dei Tintori, Monza, ITA
7Department of Medicine and Surgery, University Milano-Bicocca, Monza, Italy
8Hematology Department, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
9Hematology Division and Bone Marrow Unit, Fondazione IRCCS San Gerardo dei Tintori, Monza, MB, Italy
10Department of Medicine and Surgery, University of Milano - Bicocca, Monza, ITA
11Department of Pathology, University Milan Bicocca, Monza, ITA
12Hematology Division and Bone Marrow Unit, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
13IRCCS San Raffaele Scientific Institute, Milano, ITA
14Department of Medicine and Surgery, University of Milano Bicocca, Monza, ITA
15Biomedical Research in Melanoma-Animal Models and Cancer Laboratory, Vall D'Hebron Research Institute (VHIR), Vall D'Hebron Hospital Barcelona-Uab, Barcelona, ESP
16Department of Pathology, A.O.U Città della Salute e della Scienza, Torino, Italy
17Department of Molecular Medicine, Az. Policlinico Umberto I, Sapienza University of Rome, ROME, ITA
18Department of Translational and Precision Medicine, Hematology-Sapienza University, Rome, Italy
19Department of Clinical and Biological Sciences, University of Turin, Orbassano, ITA
20Clinical Trial Center, Translational Research and Phase I Unit, ASST Spedali Civili di Brescia, Brescia, BS, Italy
21Clinical Trial Center, Translational Research and Phase I Unit, ASST Spedali Civili di Brescia, Brescia, Italy
22Division of Hematology and Bone Marrow Transplant, AOU Policlinico “Rodolico – San Marco”, Catania, Italy
23Centro Tettamanti, IRCCS San Gerardo dei Tintori, MONZA, ITA
24Centro Tettamanti, IRCCS San Gerardo dei Tintori, Monza, ITA
25Centro Tettamanti, Fondazione IRCCS San Gerardo dei Tintori, Monza, Italy
26School of Medicine and Surgery, University of Milan-Bicocca, Monza, Italy
27Department of Pathology, University of Milano-Bicocca, Monza, Italy
28Children's Hospital Boston, Boston, MA
29Hematology Division and Bone Marrow Unit, IRCCS Foundation San Gerardo dei Tintori, Monza, Italy., Monza, Italy

Somatic SETBP1 mutations are found in various myeloid disorders covering both myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS).

To characterize the early steps of SETBP1-mediated leukemogenesis, we generated a conditional mouse model expressing SETBP1G870S mutant in the entire hematopoietic tissue through Cre-mediated recombination driven by the Vav1 promoter.

In all mice signs of a hematological disease appeared between 30 and 90 days: longitudinal analysis revealed accumulation of white blood cells (WBC) in virtually all heterozygous SETBP1G870S mice, with a marked imbalance between the lymphoid and myeloid lineages in favor of the latter, and an increase in mature myeloid cells in absence of circulating blasts or non-segmented myeloid precursors. Kaplan-Meier analysis revealed a dramatic decrease in event-free survival in SETBP1G870S mice.

BM histology showed overt myeloid hyperplasia with fibrosis and no evidence of dysplasia except for the megakaryocytic lineage. Exploration of visceral organs highlighted the presence of severe hepatosplenomegaly with massive infiltration by myeloid elements, disruption of normal tissue architecture, and signs of extramedullary hematopoiesis.

Single-cell RNA-sequencing (scRNA) on BM Lin- cells identified Mecom, Setbp1 and Hoxa9 among the top upregulated genes in SETBP1G870S precursors.

Pseudotime analysis of scRNA data revealed the presence of 261 spatially autocorrelated genes. Of them 158 were also differentially expressed in SETBP1G870S vs control mice. Spi1, encoding for the master regulator of hematopoietic differentiation PU.1, was significantly upregulated in SETBP1G870S precursors. PU.1 promotes the maturation of bone marrow early precursors towards the granulocytic/monocytic lineages by directly impairing the transcription of Gata2 and Gata1. In line with these data, Gata2 and Gata1 expression was profoundly suppressed in the early myeloid precursors of SETBP1G870S mice, which associated with the down-modulation of markers of the erythroid lineage, such as the Carbonic Anhydrase 1, in the MEP differentiation branch.

Our mouse model recapitulates many clinical features of primary myelofibrosis (PMF). As up to 10% of PMF are triple-negative for the classical JAK2, CALR, and MPL mutations (TN-PMF), we set out to assess SETBP1 mutations in this context. We analyzed 36 TN-PMF patients by exome sequencing. In 29 we did not find any evidence of somatic mutations; in the remaining 7 (7/36; 19.4%) high VAF SETBP1 degron mutations were identified. In two patients SETBP1 was found as a single somatic variant, while in the others it coexisted with ASXL1, NRAS, TET2, SRSF2, RIT1, CBL or CSF3R mutations. Notably, SETBP1 mutations were the only shared alterations among all seven patients. A markedly reduced overall survival was observed for SETBP1 positive patients, with a median survival time of 24 months (median survival not reached at 60 months for SETBP1 negative patients).

To dissect the clonal architecture of SETBP1 positive TN-PMF at single-cell resolution, we applied single-cell targeted DNA sequencing on 3 SETBP1-mutated TN-PMF samples using the Tapestri technology, showing that, as opposite to MDS/MPN, in all TN-PMF cases SETBP1 is a very early clonal event.

Therefore, our study suggests a clear partition of TN-PMF into two groups, the first one characterized by oncogenic, high VAF SETBP1 mutations accompanied by other oncogenic variants and poor prognosis and the other one characterized by no evidence of an active clonal process or of driver oncogenic events and much lower aggressiveness.

SETBP1 positive TN-PMF disorders lie in a gray zone comprised between the MPN and MPN/MDS boundary. In MDS/MPN, SETBP1 mutations are often found as mid or late events. In contrast, we show here that in TN-PMF SETBP1 appears to be one of the earliest hits, therefore highlighting a potentially relevant biological difference occurring in the two subsets. In this context the presence of early SETBP1 mutations seems to promote the occurrence of a myeloid disorder characterized by the triad: leukocytosis without differentiation block or dysplasia, BM fibrosis and progressive splenomegaly, hence recapitulating many clinical features of overt or prefibrotic/early PMF and resembling the SETBP1 mouse model.

Disclosures: Breccia: Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; BMS: Honoraria; AOP: Honoraria; AbbVie: Honoraria. Roccaro: Italian Foundation for Cancer Research; Transcan2-ERANET; AstraZeneca: Research Funding; Amgen, Celgene, Janssen. Takeda: Consultancy. Stagno: Incyte, Novartis, Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

*signifies non-member of ASH