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892 Inhibition of PRMT1 Mediated FLT3 Arginine Methylation As a Potent Therapeutic Strategy for MLL-r ALL

Program: Oral and Poster Abstracts
Type: Oral
Session: 605. Molecular Pharmacology, Drug Resistance—Lymphoid and Other Diseases: Molecular Mechanisms in Leukemic Drug Resistance
Hematology Disease Topics & Pathways:
Biological, ALL, Leukemia, Diseases, Therapies, Biological Processes, enzyme inhibitors, Lymphoid Malignancies, hematopoiesis
Monday, December 3, 2018: 5:15 PM
Room 29C (San Diego Convention Center)

Yinghui Zhu, PhD1*, Xin He, PhD2*, Haojie DONG, PhD1*, Jie Sun, PhD1*, Hanying Wang, PhD1*, Lei Zhang, PhD3*, Yunan Miao, PhD3*, Jian Jin, PhD4*, Yudao Shen, PhD4*, Jianjun Chen, PhD5, Markus Muschen, MD6,7, Chun-Wei Chen, PhD8, Marina Y. Konopleva, MD, PhD9, Weili Sun, MD, PhD10, Bin Zhang, PhD1*, Ya-Huei Kuo, PhD1, Nadia Carlesso, MD, PhD1, Guido Marcucci, MD1 and Ling Li, PhD11

1Department of Hematological Malignancies Translational Science, Gehr Family Center for Leukemia Research, Hematologic Malignancies and Stem Cell Transplantation Institute, Beckman Research Institute, City of Hope Medical Center, Duarte, CA
2Department of Hematological Malignancies Translational Science, Gehr Family Center for Leukemia Research, Hematologic Malignancies and Stem Cell Transplantation Institute, Beckman Research Institute, City of Hope Medical Center, Monrovia, CA
3Translational Biomarker Discovery Core, Beckman Research Institute, City of Hope Medical Center, Duarte, CA
4Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY
5Department of Systems Biology, Beckman Research Institute, City of Hope Medical Center, Monrovia, CA
6Department of Systems Biology, City of Hope Comprehensive Cancer Center, Monrovia CA, Monrovia, CA
7Department of Systems Biology, City of Hope Comprehensive Cancer Center, Monrovia CA, San Francisco, CA
8Department of Systems Biology, Beckman Research Institute, City of Hope, Duarte, CA
9Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX
10Department of Pediatrics, City of Hope Medical Center, Duarte, CA
11Department of Hematological Malignancies Translational Science, Gehr Family Center for Leukemia Research, Hematologic Malignancies and Stem Cell Transplantation Institute, Beckman Research Institute, City of Hope Medical Ctr., Beckman Res. Institute, Duarte, CA

Mixed-lineage leukemia-rearranged (MLL-r) ALL, seen in 70% of infant ALL, has a dismal prognosis compared to those with wild type MLL1 gene. Transcriptional profiling has identified Fms-like receptor tyrosine kinase 3 (FLT3) as one of the most significantly upregulated genes in MLL-r ALL. The highly expressed FLT3 protein is activated by the autocrine ligand, making the kinase a therapeutic target. FLT3 tyrosine kinase inhibitors (TKIs) such as PKC412, although effective in kinase inhibition, partially impair survival of MLL-r ALL cells and clinical trial results are not promising, promoting us to ask whether FLT3 regulates the ALL cells survival also through a kinase-independent mechanism.

Herein, we report the finding of dimethylated arginines on FLT3, detected through mass spectrometry analysis of a MLL-r ALL specimen and a MLL-r ALL line SEM. The most conserved and enriched of dimethylated arginines are residues R972/R973. Using home-made arginine methylation (R-Me) antibody, we found that PRMT1, which is responsible for most type I arginine methyltransferases activity, catalyzes FLT3 methylation. Immunoblot (IB) analysis validated the expression of FLT3 R-Me in MLL-r ALL samples (6 out of 6) and MLL-r ALL lines (4 out of 4). Analysis of the GEO dataset (GSE13204) revealed that PRMT1 mRNA levels are increased in MLL-r ALL relative to normal cells (MLL-r, n=70 vs. normal, n=73, p<0.0001). We studied FLT3 R-Me biological function using two approaches that specifically blocked FLT3 methylation levels: cells expressing FLT3 methylation deficient construct (R972/973K, arginine [R] to lysine [K]) exhibited reduced survival (BaF3: FLT3-WT 98.5±0.11% vs. R972/973K 71.5±0.53%, p=0.0004); knockdown of PRMT1 in SEM cells also had an inhibitory effect (siCtrl 95.1±0.1% vs. siPRMT1 74.7±0.5%, p=0.0007). Moreover, the type I arginine methyltransferase inhibitor MS023 (5 µM) treatment markedly induced apoptosis of primary ALL cells but spared normal counterparts from healthy donors (ALL: vehicle 10.4±0.4% vs. MS023 23.7±0.8%, n=4; p<0.0001; normal CD19+: 8.3±0.3% vs. 8.2±0.1%, n=3, p=0.86). Interestingly, inhibition of FLT3 methylation decreased FLT3 phosphorylation at tyrosine 969 (Y969) but not Y589/591 or Y842. Expression of R972/973K decreased FLT3 downstream signaling like phospho-STAT5 and -AKT to a greater extent than that of Y969F mutant (Y to phenylalanine [F] substitution, mimics loss of Y phosphorylation). Next, FLT3 WT, R972/973K or Y969F transduced primary MLL-r ALL cells were transplanted into NSGS mice for analysis of leukemia development (n=6/group). Mice transplanted with FLT3 Y969F MLL-r ALL had longer survival relative to FLT-WT injected animals (p=0.0031), and the median survival was further extended in mice injected with R972/973K mutant compared with FLT3 Y969F MLL-r ALL (p=0.0007). Additionally, PKC412 treatment alone did not alter FLT3 R-Me, and high FLT3 methylation level in SEM cells was not affected by FLT3 ligand stimulation, confirming that the function of R-Me is independent of FLT3 phosphorylation. Importantly, we observed that the combination of MS023 with PKC412 significantly induced a higher rate of apoptosis in primary MLL-r ALL cells compared with each drug alone (control, 10±0.43%, MS023, 21.1±1.2%, PKC412, 21.5±0.11%, combination, 39.8±2.9%, PKC412 vs combination, p<0.01, n=4). We further tested the effects of in vivo administration of MS023 plus PKC412 on primary MLL-r ALL cells xenografted in NSGS mice. Following engraftment >1% in peripheral blood, mice were subdivided into four groups and treated with vehicle, PKC412 (100 mg/kg, i.g.), MS023 (80 mg/kg, i.p, bid), or the combination (n=7/group) for 4 weeks. The BM tumor burden of CD45+ CD19+ cells was reduced in single drug-treated mice cohorts, with further reduction after combination treatment (vehicle, 94.4±0.5%, PKC412, 50.2±6.3%, MS023, 55.6±4.5%, combination, 30.7±4.9%, PKC412 vs. combination, p<0.001). Secondary transplantation of BM cells from mice receiving combination treatment resulted in significantly reduced BM engraftment at 16 weeks compared to PKC412 treatment alone (PKC412, 62.2±4.9%, combination, 8.4±5.1%, n=5, p<0.0001), indicating reduced leukemia initiating capacity.

Our results support further exploring the molecular function of FLT3 R-Me. We will determine whether PRMT1 and FLT3 methylation are potential druggable targets in MLL-r ALL.

Disclosures: Konopleva: Stemline Therapeutics: Research Funding.

*signifies non-member of ASH