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1785 Loss of Function of SETD2 Tumor Suppressor in Chronic Myeloid Leukemia (CML) Progenitors Fosters Genomic Instability and Enhances Clonogenic Potential

Program: Oral and Poster Abstracts
Session: 631. Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Translational Research, CML, Chronic Myeloid Malignancies, Diseases, Myeloid Malignancies, Biological Processes, molecular biology, Technology and Procedures
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Sara De Santis1*, Manuela Mancini, PhD1*, Cecilia Monaldi2*, Gabriele Gugliotta, MD3*, Samantha Bruno, PhD1*, Fausto Castagnetti, MD, PhD1,3, Miriam Iezza, MD1*, Marco Cerrano, MD4*, Alessandra Iurlo, MD, PhD5*, Sara Galimberti, MD, PhD6,7*, Serena Balducci6*, Fabio Stagno, MD, PhD8, Michele Cavo, MD1,3* and Simona Soverini, PhD1

1Dipartimento di Scienze Mediche e Chirurgiche (DIMEC), Università di Bologna, Bologna, Italy
2Dipartimento di Scienze Mediche e Chirurgiche (DIMEC), Università di Bologna, Bologna, AL, Italy
3IRCCS Azienda Ospedaliero–Universitaria di Bologna, Istituto di Ematologia "Sèragnoli", Italy, Bologna, Italy, Bologna, Italy
4Division of Hematology, Department of Oncology, A.O.U. Città della Salute e della Scienza di Torino, Torino, Italy
5Hematology Division, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
6Department of Clinical and Experimental Medicine, Hematology, University of Pisa, Pisa, Italy
7UO Ematologia, AOU Santa Chiara, Pisa, Italy
8Division of Hematology and Bone Marrow Transplant, AOU Policlinico “Rodolico – San Marco”, Catania, Italy

BACKGROUND AND AIMS – Genomic instability is a hallmark of chronic myeloid leukemia (CML) cells since the chronic phase (CP) of the disease, and results in BCR::ABL1 mutations and/or additional genetic and genomic aberrations that may drive resistance to tyrosine kinase inhibitors (TKIs) and progression to blast crisis (BC). Genomic instability is also a feature of CML stem and progenitor cells and may contribute to their persistence. The SETD2 tumor suppressor codes for a protein that trimethylates histone H3 at lysine 36 (H3K36me3). In solid tumors, SETD2 loss of function has been shown to impair H3K36me3-mediated recruitment of DNA damage response components. We have recently reported that non genomic loss of function of SETD2 is a feature of BC CML and results from premature proteasome-mediated degradation of the SETD2 protein triggered by Aurora kinase A phosphorylation and MDM2 ubiquitination. In the present study, we aimed to assess SETD2/H3K36me3 status in CD34+ progenitors of CP CML patients (pts) and whether SETD2/H3K36me3 deficiency may play a role in genomic instability in CML models.

METHODS – Western blotting (WB) was used to assess SETD2 protein expression and H3K36me3 as a surrogate marker of SETD2 function in the CD34+ cell fraction isolated from the bone marrow of 20 newly diagnosed CP CML pts and from a pool of healthy donors (HD). SETD2 forced expression in CD34+ progenitors from newly diagnosed CP CML pts and in the SETD2-deficient KCL22 cell line was performed by nucleofection. SETD2 knock-down in the SETD2-proficient LAMA84 cell line was performed by RNAi. Clonogenic capacity was evaluated by clonogenic assays. Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K36me3 was performed on an Illumina HiSeq2000 with a min 50 million 150-bp single-end reads per replicate. High resolution karyotyping wias perfomed with Cytoscan HD arrays. DNA damage and DNA repair activation were assessed in primary samples and cell lines by WB and immunofluorescence (IF) using antibodies specific for phospho-H2AX, mismatch repair (MMR) and homologous recombination repair (HR) proteins.

RESULTS – WB demonstrated a marked down-modulation of SETD2 expression, paralleled by H3K36me3 deficiency, in the CD34+ cells of all newly diagnosed CP CML pts as compared to the total mononuclear fraction and to CD34+ cells from HDs. To investigate whether SETD2 loss affects the activation and proficiency of HR and MMR, we used chronic exposure to UV rays or a single exposure to hydrogen peroxide (1mM for 30 and 60 min). Both induced DNA damage in SETD2 siRNA-depleted LAMA84 cells. Compared to parental cells, cells silenced for SETD2 failed to activate the ATM-dependent repair pathway. Moreover, we found that ATM inactivation prevents H2AX foci formation, associated with a loss of RAD51 and RAD54 (HR) and MSH6 (MMR) repair foci. To confirm the hypothesis that SETD2 is a tumor suppressor implicated in maintaining genomic stability in CML, we transfected SETD2-deficient KCL22 cells with an ectopic SETD2 plasmid. SETD2 forced expression was able to restore DNA damage response, as demonstrated by WB and IF detection of ATM, p95 and H2AX phosphorylation, BRCA1, BRCA2 and CtIP expression and finally RAD51 and RAD54 localization on HR repair foci observed after UV or hydrogen peroxide exposure. High-resolution karyotyping after DNA damage showed increased rate of DNA breakpoints in SETD2-deficient cells, preferentially occuring at loci where H3K36me3 marks were lost as assessed by ChiP-seq.

In line with the effects observed in cell line models, forced expression of SETD2 in CD34+ cells from 5 CP CML pts was found to restore proliferation control, since a >50% reduction in clonogenic potential was observed after nucleofection.

CONCLUSIONS – Our findings demonstrate that SETD2 is a bona fide tumor suppressor in CML progenitors and establish a functional link between SETD2 loss of function and genomic instability. SETD2 inactivation may thus play a pivotal role in the harmful cascade of events that may foster drug resistance and ultimately lead to disease progression. Since SETD2 loss of function in CML is mediated by post-translation mechanisms, hence is reversible, therapeutic strategies aimed at interfering with these mechanisms may restore proliferation control and interrupt this cascade.

Supported by AIRC IG 2019 (23001) and by Italian Ministry of Health, “Bando Ricerca Finalizzata 2016”, project GR-2016-02364880.

Disclosures: Castagnetti: Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Cerrano: Insight Novartis Servier Abbvie Janssen Jazz Astellas Italfarmaco: Honoraria. Iurlo: Novartis, Pfizer, Incyte, BMS, GSK, AOP Health: Honoraria. Galimberti: Abbvie, Janssen, Novartis, Roche, Jazz, Astra Zeneca, Pfizer, Incyte: Speakers Bureau. Stagno: Incyte, Novartis, Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo: Adaptive: Honoraria; Roche: Honoraria; Sanofi: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Honoraria; Takeda: Honoraria; GlaxoSmithKline: Honoraria; Celgene/Bristol Myers Squibb: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau.

*signifies non-member of ASH