Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in AML

Abstract

Background: NUP98-NSD1 positive AML is a poor prognostic subgroup within pediatric and adult AML (Thol et al., 2013). However, targeted therapeutics for these AML patients are not available to date. As a result of the NUP98-NSD1 fusion, NSD1 causes H3K36 hypermethylation of HOXA genes, which contributes to myeloid progenitor cell immortalization and results in AML (Wang et al., 2007). Therefore, we hypothesized that inhibition of the methyltransferase activity of NSD1 could be an effective treatment strategy for NUP98-NSD1 AML patients. Here, we assessed the efficacy of NSD1 inhibitor suramin and NUP98-NSD1-directed siRNA-containing lipid nanoparticles (LNP) in a preclinical patient-derived xenograft (PDX) model of NUP98-NSD1 leukemia. Methods: A NUP98-NSD1 positive AML patient was screened through nested PCR and Sanger sequencing. Bone marrow cells from this patient were serially transplanted into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice to establish a NUP98-NSD1 PDX model. During serial transplantations, an alternative NUP98-NSD1 fusion gene appeared. Flow-cytometry was used to check the engraftment and immunophenotype of engrafted cells. Effective siRNAs against each of the two fusion genes were developed. The microfluidic mixing technology, Nanoassemblr was used to package siRNA into LNPs and the Zetasizer was used to characterize them. Fifteen days after transplantation, suramin and solvent treatment were initiated in 9 mice per group with 10 mg/kg suramin (2 days/week for 10 weeks). For LNP treatment, treatment was initiated 3 weeks after transplantation with a once daily dose of 2.5 mg/kg on days 1-3 and then every third day thereafter for a total of 17 injections. Results: Besides the NUP98-NSD1 translocation, our patient had a FLT3-ITD mutation and a normal karyotype. In our PDX model, the engraftment of human leukemia cells reached up to 90% after ten weeks of transplantation. In successive transplantations up to the 6th generation, the NUP98-NSD1 fusion was confirmed in leukemic cells, supporting the importance of the fusion for leukemia development and stability of the model. In later transplantations, a minor clone of NUP98-NSD1 was identified. A high blast count, high WBC count, increased spleen weight, and a low hemoglobin and platelet count at death demonstrated the development of acute leukemia. High expression of myeloid markers (e.g. CD33, 99%, N=9) and negligible expression of lymphoid markers (CD3, 2%; CD19, 2%; N=9) confirmed acute myeloid leukemia. In the suramin treatment study, the mean human leukemic cell engraftment was similar between the control and treatment groups at the start of treatment (0.51%, N=9 and 0.71%, N=9, respectively), but was lower in suramin treated mice after 4 and 8 weeks of treatment (4 weeks: CTRL, 4,8%; suramin, 2,66%, P=0.1; 8 weeks: CTRL, 87,3%; suramin: 66,5%, P=0.016). No significant effect was seen on the immunophenotype of suramin and control treated leukemia cells. Suramin treatment significantly prolonged the median survival of mice compared to control mice (126 vs 114 days after transplantation, P=0.008). To establish the siRNA-LNP treatment, we identified one siRNA against each NUP98-NSD1 clone that reduced expression levels by 78% and 89.5% in the major and minor clones, respectively. The effective siRNAs were modified to increase their in vivo stability and were packaged into LNPs and used in vivo. We started the treatment when the engraftment was similar in both control LNP and NUP98-NSD1 LNP groups (0.93%, N=7 and 1.25%, N=6, respectively). After 3 weeks of treatment, LNP uptake was 99.3% and 99.2% in the CTRL LNP and NUP98-NSD1 LNP groups, respectively. The mean engraftment was lower in NUP98-NSD1 LNP mice after 5 and 8 weeks of treatment (5 weeks: CTRL LNP, 15%; NUP98-NSD1 LNP, 4.6%, P= 0.08; 8 weeks: CTRL LNP, 94.8%; NUP98-NSD1 LNP, 55.83%, P=0.007). Importantly, the NUP98-NSD1 siRNA-LNP treated mice showed a significant survival benefit compared to CTRL siRNA-LNP treated mice (106 vs 82 days after transplantation, P=0.02). Conclusions: In summary, our findings demonstrate that targeted inhibition of NUP98-NSD1 either through siRNA-LNP or suramin delays leukemia development in vivo and prolongs the survival of mice carrying a NUP98-NSD1 positive AML. Our results provide the rationale for the evaluation of NSD1 methyltransferase inhibitors and siRNA-LNP formulations in NUP98-NSD1 positive AML. Disclosures Heuser: Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding.

NUP98 Rearrangements in AML: Molecular Mechanisms and Clinical Implications

“…Furthermore, the development of patient-derived xenograft (PDX) models of NUP98 fusions recapitulates the disease well and is an effective method to track patient cells' response to different drugs. We currently have PDX models for only a few NUP98 rearrangements, but we will need to establish PDX models for all common NUP98 rearrangements in the near future [26,158,159]. In different NUP98 fusion mouse models, the siRNA-LNP formulations, CDK6 inhibitor, and menin inhibitor show promising antileukemic activity.…”

Drug Resistance Mechanisms of Acute Myeloid Leukemia Stem Cells

“…A recent study reported that targeting inhibitor of NSD1, BT5, reduced the expression levels of HOX cluster and MEIS1 and impaired the colony formation of primary AML cells ( 172 ). And inhibition of NUP98-NSD1 reduced leukemia burden and prolonged survival in NUP98-NSD1 patient-derived xenograft model ( 172 , 173 ).…”

Straight to the Point—The Novel Strategies to Cure Pediatric AML

“…This rearrangement, initially described in M7 pediatric AML, occurs in 2% of all childhood cases, and it is associated with a poor prognosis with 5-year OS rate of around 33% [34][35][36]. Sagarajit Mohanty et al [106] analyzed the synergistic effect of NUP98::NSD1 and NRASG12D using an in vivo mouse model. To demonstrate the leukemic potential of NRASG12D, NUP98::NSD1, and NUP98::NSD1 + NRASG12D in vivo, they transplanted transduced mouse bone marrow cells into mice.…”

“…Reported data might suggest that targeted inhibition of the fusion might be a potential treatment in NUP98::NSD1-positive AML patients. This appears to be a promising area for further clinical research [106]. In other preclinical studies, Johannes Schmoeller et al [107] investigated the effectiveness of CDK4/CDK6 inhibition in in vivo and in vitro models of NUP98-fusion AML.…”