Mutant SETBP1 enhances NRAS-driven MAPK pathway activation to promote aggressive leukemia

Abstract

Mutations in SET-binding protein 1 (SETBP1) are associated with poor outcomes in myeloid leukemias. In the Ras-driven leukemia, juvenile myelomonocytic leukemia, SETBP1 mutations are enriched in relapsed disease. While some mechanisms for SETBP1-driven oncogenesis have been established, it remains unclear how SETBP1 specifically modulates the biology of Ras-driven leukemias. In this study, we found that when co-expressed with Ras pathway mutations, SETBP1 promoted oncogenic transformation of murine bone marrow in vitro and aggressive myeloid leukemia in vivo. We demonstrate that SETBP1 enhances the NRAS gene expression signature, driving upregulation of mitogen-activated protein kinase (MAPK) signaling and downregulation of differentiation pathways. SETBP1 also enhances NRAS-driven phosphorylation of MAPK proteins. Cells expressing NRAS and SETBP1 are sensitive to inhibitors of the MAPK pathway, and treatment with the MEK inhibitor trametinib conferred a survival benefit in a mouse model of NRAS/SETBP1-mutant disease. Our data demonstrate that despite driving enhanced MAPK signaling, SETBP1-mutant cells remain susceptible to trametinib in vitro and in vivo, providing encouraging preclinical data for the use of trametinib in SETBP1-mutant disease.

Figure 1.. SETBP1^D868N enhances the proliferation of NRAS^G12D and PTPN11^E76K hematopoietic progenitors.

(A) Representative images of colony forming unit (CFU) assays show that SETBP1^D868N enhances the colony forming capacity of PTPN11^E76K. (B) Quantification showing that PTPN11^E76K synergizes with SETBP1^D868N to produce significantly more colonies than with PTPN11^E76K alone. Serial replating of cells co-transfected with PTPN11^E76K and SETBP1^D868N. 5,000 cells were sorted into methocult for the first plating and 10,000 cells were harvested, washed and re-plated for the subsequent platings. (C) Representative images show an enhancement of colony forming potential in cells transduced with both NRAS^G12D and SETBP1^D868N mutations. While NRAS^G12D alone produces some small dense colonies, the combination of NRAS^G12D and SETBP1^D868N results in an increased number of colonies that were generally larger than with NRAS^G12D alone. (D) Quantification of colony number showing that when combined with SETBP1^D868N mutation, the NRAS^G12D mutation produces significantly more colonies than with NRAS^G12D alone. For this assay, 1,500 cells were sorted into methocult for the first plating and 10,000 cells were harvested, washed and re-plated for the subsequent platings. Enhanced serial replating of NRAS^G12D and SETBP1^D868N expressing progenitors relative to those expressing NRAS^G12D alone. Statistical significance is represented as *p<0.05, **p<0.01. (E) 5,000 lineage-depleted mouse bone marrow cells expressing SETBP1^D868N and/or NRAS^G12D with the appropriate retroviral control vectors were transplanted into lethally irradiated mice with 200,000 carrier bone marrow cells. The median survival in mice with BOTH oncogenes (SETBP1^D868N/NRAS^G12D) was 20 days, compared to 149.5 days with SETBP1 alone (SETBP1^D868N/Empty). Mice receiving cells expressing NRAS alone (Empty/NRAS^G12D) did not reach their median survival by 165 days. Significance was determined by logrank (Mantel-Cox test), with the threshold for significance of p-value < 0.0083. Number of mice per group were as follows (N_EMPTY=6; N_NRAS=7; N_SETBP1=4; N_BOTH=5). (F) At 20 days post-transplant (the median survival time for mice with BOTH mutations together (SETBP1^D868N/ NRAS^G12D)), a marked elevation of white blood cells is seen relative to all other groups. (G) Peripheral complete blood counts were monitored over time. Mice expressing mice with BOTH oncogenes (SETBP1^D868N/ NRAS^G12D) developed high WBC counts in the first three weeks, while mice with SETBP1 only (SETBP1^D868N/Empty) began to develop high WBC counts after 17 weeks. (H) RNAseq differential expression analyses were performed on transduced lineage depleted murine bone marrow cells expressing empty vector (Empty/Empty), SETBP1 (SETBP1^D868N/Empty), NRAS (Empty/ NRAS^G12D) or BOTH oncogenes (SETBP1^D868N/NRAS^G12D). The Venn diagram shows the number of genes that are differentially expressed relative to the empty vector control for SETBP1, NRAS and BOTH (logFC +/− 1.5, adj p-value < 0.05). There were 402 differentially genes at the intersection of NRAS and BOTH, and 399 genes that were differentially expressed only in the BOTH group. Enrichr analysis of these genes that are only differentially expressed with BOTH oncogenes showed upregulation of inflammatory and Ras/MAPK pathways. (I) Unsupervised clustering of differentially-expressed genes (logFC +/− 1.5, adj p-value < 0.05). Cluster 1 shows a strong MAPK signature in genes upregulated by both SETBP1 and NRAS. Cluster 4 is enriched for KEGG pathways associated with myeloid differentiation.

Figure 2.. SETBP1^D868N enhances MAPK signaling driven by NRAS^G12D.

(A) Immunoblot analysis of NRAS^G12D/ SETBP1^D868N expanded hematopoietic progenitors reveals increased activation of MAPK and mTOR signaling relative to a normal marrow control. (B) A chemical screen with commercially available inhibitors was performed on our novel NRAS^G12D/SETBP1^D868N cell line to identify essential cell growth and survival pathways, and the cells were found to be highly sensitive to Raf/MEK/ERK inhibitors (black). (C) Immunoblot analysis of 293T17 cells transiently transfected with and empty vector alone, NRAS^G12D, SETBP1^D868N or the combination of both genes. An empty vector control is used to control for the total amount of plasmid transfected. Co-transfection with NRAS^G12D and SETBP1^D868N increases the phosphorylation of ERK and MEK above NRAS^G12D alone. Deletion of the SET-binding domain from SETBP1^D868N (SETBP1^delSET) does not reduce MEK/ERK activation relative to full length SETBP1^D868N. (D) To validate the efficacy of identified inhibitors against the NRAS^G12D/SETBP1^D868N cells, a 7-point dose response curve (0–500nM) with a 1:3 fixed molar ratio of each of the top agents was performed, and a percent viability calculated relative to untreated cells after 72 hours. Rapamycin had sub-nanomolar efficacy. (E) Trametinib had sub-nanomolar efficacy. (F) The efficacy of FTY720 and Rigosertib was evaluated in our cell line using an 11-point curve (0–1000nM) with a 1:3 fixed molar ratio. (G) To evaluate the efficacy of trametinib in vivo, 100,000 NRAS^G12D/SETBP1^D868N-mutant cells were retro-orbitally injected into C75BL/6J mice without irradiation. Beginning at day 13 (dotted vertical line), mice were given once-daily treatment of either DMSO (N=6), 10 mg/kg rapamycin (N=7) or 1 mg/kg trametinib (N=7). Median survival in mice receiving the DMSO control treatment was 19.5-days post-transplant compared 42 days with trametinib (p=0.0007). Significance was determined by logrank (Mantel-Cox test). Mice treated with rapamycin had a median survival of 21 days. (H) At Day 21, the disease burden was markedly higher in the peripheral blood of DMSO-treated mice relative to trametinib-treated mice. (I) WBC count over time as measured by automated CBC. (J) The efficacy of trametinib in human blood cells was evaluated using two CMML patient samples with NRAS^G12D/SETBP1^{G870S or I871S}. 100,000 blood cells per well were plated in triplicate in a CFU assay with increasing doses of trametinib (50, 100, 200nM).