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1726 Aurora Kinase a/MDM2-Mediated SETD2 Loss of Function in Chronic Myeloid Leukemia Patients in Blast Crisis Induces Genetic Instability and Can be Therapeutically Targeted

Program: Oral and Poster Abstracts
Session: 631. Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy: Poster I
Hematology Disease Topics & Pathways:
Adult, apoptosis, Biological, Diseases, Therapies, CML, Biological Processes, enzyme inhibitors, DNA repair, Study Population, Myeloid Malignancies, TKI, pathogenesis
Saturday, December 1, 2018, 6:15 PM-8:15 PM
Hall GH (San Diego Convention Center)

Manuela Mancini, PhD1*, Sara De Santis1*, Cecilia Monaldi1*, Luana Bavaro2*, Margherita Martelli2*, Fausto Castagnetti3, Gabriele Gugliotta, MD, PhD4, Gianantonio Rosti4*, Maria Chiara Fontana, MSc5*, Elisa Dan6*, Barbara Sinigaglia6*, Alessandra Iurlo7*, Nicola Orofino8*, Elisabetta Abruzzese9, Marzia Salvucci10*, Patrizia Pregno11*, Antonella Gozzini12*, Monica Crugnola, MD13*, Francesco Albano, MD14*, Massimiliano Bonifacio15*, Elisabetta Calistri, MD16*, Mario Tiribelli17*, Gianni Binotto, MD18*, Annalisa Imovilli19*, Elena Trabacchi, MD20*, Sara Galimberti21*, Claudia Baratè22*, Elena Tenti23*, Michele Baccarani, MD24, Giovanni Martinelli25*, Michele Cavo, MD26* and Simona Soverini, PhD27

1Department of Experimental Diagnostic and Specialty Medicine - DIMES Institute of Hematology "L. e A. Seràgnoli", University of Bologna, Bologna, Italy
2Hematology/Oncology "L. e A. Seràgnoli", University of Bologna, Bologna, Italy
3Institute of Hematology "L. e A. Seràgnoli", DIMES, University of Bologna, Bologna, Italy
4Institute of Hematology "L. and A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, Bologna, Italy
5Institute of Hematology "L. and A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy
6Department of Experimental, Diagnostic and Specialty Medicine - DIMES, Institute of Hematology "L. and A. Seràgnoli", Bologna, Italy
7Hematology Division, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy
8UO Ematologia, Fondazione IRCCS Ca' Granda - Ospedale Policlinico, Milano, Milano, Italy
9Hematology Unit, S. Eugenio Hospital, Roma, Italy
10Azienda Azienda USL di Romagna, Ravenna, Ravenna, Italy
11Hematology Unit, Az Ospedaliero Universitaria Città' della Salute e della Scienza, Torino, Italy
12Divisione di Ematologia-Policlinico Careggi di Firenze, Firenze, ITA
13Department of Medicine and Surgery, University of Parma, Parma, Italy
14University of Bari, Department of Emergency and Organ Transplantation (D.E.T.O.), Hematology Section, Bari, Italy
15Department of Medicine, Section of Hematology, Stem Cell Research Lab, University of Verona, Verona, Italy
16UO Hematology, Ca Foncello Hospital, Treviso, Italy
17Division of Hematology and BMT, Department of Medical Area, University of Udine, Udine, Italy
18Hematology and Clinical Immunology, Department of Medicine, Padua School of Medicine, Padova, Italy
19Hematology Unit, Arcispedale Santa Maria Nuova IRCCS, Reggio Emilia, Italy
20Hematology and BMT Unit, Department of Hematology and Oncology, Guglielmo da Saliceto Hospital, Piacenza, Italy
21Hematology Department, University of Pisa, Pisa, Italy
22UO Ematologia - AOU Pisana - Pisa, Pisa, Italy
23)Department of Experimental, Diagnostic and Specialty Medicine - DIMES, Institute of Hematology "L. and A. Seràgnoli", Bologna, Italy
24Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Institute of Hematology and Medical Oncology "L. & A. Seràgnoli", Bologna, Italy
25Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy, Meldola, Italy
26Institute of Hematology "L. and A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, "S. Orsola-Malpighi" Hospital, Bologna, Italy
27Institute of Hematology "L. and A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, "S. Orsola-Malpighi" Hospital, Castel Maggiore, Bologna, Italy

The SETD2 protein is a histone methyltransferase that specifically catalyzes the trimethylation of Lysine 36 on histone H3 (H3K36me3). SETD2/H3K36me3 are implicated in transcript elongation and splicing, DNA repair, chromosome segregation. SETD2 gene deletions and/or mutations (mostly frameshift or nonsense) have been reported in solid tumors (clear cell renal cell carcinoma, bladder cancer, lung cancer, melanoma, endometrial cancer) and in acute leukemias.

Using a Western Blotting (WB) approach to screen for SETD2 protein expression and for H3K36me3 levels in a relatively large cohort of 80 advanced-phase chronic myeloid leukemia (CML) patients (pts), we could detect reduced or null SETD2 and H3K36me3 in 86% of pts as compared to a pool of healthy donors and to chronic phase (CP) pts at diagnosis who achieved optimal responses to TKI, but neither mutations/deletions nor transcriptional down-regulation were the underlying causes. Inhibition of proteasome-mediated degradation in primary cells from pts with undetectable SETD2 restored H3K36me3 and led to accumulation of hyper-ubiquitinated SETD2, suggesting that a functional protein is produced but rapidly degraded. Moreover, proteasome inhibition was found to induce apoptosis and to reduce clonogenic growth. In K562 cells (SETD2/H3K36me3low), co-immunoprecipitation (co-IP) performed before and after proteasome inhibition showed accumulation of the hyper-ubiquitinated form of SETD2 bound to MDM2. MDM2 inhibition by SP-141 resulted in cytostatic effects and restored SETD2 expression and activity. Superimposable results were achieved by siRNA-mediated silencing of MDM2, suggesting that MDM2 is implicated in SETD2 reduced stability. Co-IP also showed that SETD2 interacts with Aurora Kinase A a Ser-Thr kinase frequently overexpressed in CML. We found that Aurora Kinase A phosphorylates SETD2, and both pharmacological inhibition by Danusertib and siRNA-mediated silencing rescued SETD2 expression and activity.

Next, to investigate whether SETD2/H3K36me3 loss may contribute to genetic instability, LAMA 84 (SETD2/H3K36Me3high) and K562 (SETD2/H3K36me3low) cells were studied by WB and immunofluorescence (IF) to assess phosphorylated histone 2A.X (γH2AX) and Rad51 foci in steady state conditions and after sub-lethal DNA damage by UV exposure. The same studies were performed after SETD2 silencing for 3 months. Cells with low or silenced SETD2 had significantly higher levels of γH2AX and were unable to induce homologous recombination (HR) repair after DNA damage. Clonogenic assays performed in LAMA 84 cells before and after SETD2 silencing, in K562 (SETD2/H3K36me3low) and in imatinib-resistant (IM-R) K562 cells which have lost SETD2 expression and activity, suggested that reduction of clonogenic growth after proteasomal or MDM2 inhibition is strictly dependent on SETD2 expression and functional status (Figure 1A).

First and second generation proteasome inhibitors (bortezomib, carfilzomib and ixazomib) inhibited the clonogenic potential of the mononuclear cell fraction from both CP (n=2) and blast crisis (BC) (n=4) CML pts at subnanomolar concentrations, with the extent of anti-tumor activity clearly anti-correlated with SETD2 expression and H3K36me3 levels: pts with lower SETD2 expression showed lower LD50 when compared with pts with higher SETD2 expression and H3K36me3 levels (Figure 1B). Similarly, clonogenic assays performed by administrating increasing doses of SP-141 (from 0.25 to 1.25 µM) suggested that MDM2 specific inhibition had more significant effects in BC-CML pts showing low SETD2 levels and activity as compared to BC-CML pts showing intermediate SETD2 levels and activity and to CP CML pts.

In conclusion, phosphorylation by Aurora Kinase A and ubiquitination by MDM2 contribute to SETD2 non-genomic loss of function in advanced-phase CML. Loss of SETD2/H3K36me3 is associated with increased DNA damage and impaired HR repair. Restoring physiological H3K36me3 levels may help improve the outcome of this critical subset of pts.

Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma).

Disclosures: Castagnetti: Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta: Novartis: Honoraria; Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Incyte: Honoraria. Abruzzese: Pfizer: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy. Bonifacio: Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Martinelli: Ariad/incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Roche: Consultancy. Cavo: Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini: Bristol Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.

*signifies non-member of ASH