Therapeutic targeting of preleukemia cells in a mouse model of NPM1 mutant acute myeloid leukemia

Abstract

The initiating mutations that contribute to cancer development are sometimes present in premalignant cells. Whether therapies targeting these mutations can eradicate premalignant cells is unclear. Acute myeloid leukemia (AML) is an attractive system for investigating the effect of preventative treatment because this disease is often preceded by a premalignant state (clonal hematopoiesis or myelodysplastic syndrome). In Npm1c/Dnmt3a mutant knock-in mice, a model of AML development, leukemia is preceded by a period of extended myeloid progenitor cell proliferation and self-renewal. We found that this self-renewal can be reversed by oral administration of a small molecule (VTP-50469) that targets the MLL1-Menin chromatin complex. These preclinical results support the hypothesis that individuals at high risk of developing AML might benefit from targeted epigenetic therapy in a preventative setting.

Fig. 1.. Npm1c induces self–renewal properties in myeloid progenitor cells.

(A) Hoxa9 gene expression in Npm1c, Dnmt3a and Npm1c/Dnmt3a mutant LT-HSCs,multipotent progenitors (MPPs), common myeloid progenitors (CMPs) and GMPs 16 weeks after pIpC injection (n≥3 mice per group; error bars indicate mean ± SD). Rel., relative;GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) Heatmap showing top 25 up-regulated genes in Npm1c versus WT LSK and GMPs, 4 weeks after pIpC treatment (n=3 mice per group).(C) Relative expression of Hoxa9 3 days after in vitro Cre transduction (n ≥ 4 mice per group;error bars indicate mean ± SEM). The dotted lines indicate Hoxa9 expression level from freshly isolated LSK cells and GMPs. (D) Peripheral blood percentage CD45.2 engraftment of WT and Npm1c, Dnmt3a, and Npm1c/Dnmt3a mutant GMPs sorted 4 weeks after pIpC induction, transplanted into lethally irradiated recipients. Error bars indicate mean ± SEM. (E) Summary table of GMP-transplanted mouse numbers and percentages engrafted ≥1% for >12 weeks. MIGCRE,MSCV-CRE-IRES-GFP retrovirus. ns, not significant; *P < 0.05; **P < 0.01; ***P <0.001.

Fig. 2.. Myeloid progenitors are leukemia-initiating cells in Npm1c AML.

(A) Experimental overview of secondary transplantation of long-term engrafted mutant GMPs. (B to D) Kaplan-Meyer survival analysis (B), representative spleen images (C), and spleen weights (D) of secondary transplants of MIG-CRENpm1c/Dnmt3a LT-GMPs or LSK-derived cells and MxCreNpm1c LT-GMPs (n ≥ 4 mice per group). One-way analysis of variance (ANOVA) was performed. Error bars indicate mean ± SD. *P < 0.05; ns, not significant. (E) Top 10 up-regulated genes in MIG-CRENpm1c/Dnmt3a mutant leukemic GMPs compared with WT GMPs (n = 3 mice per group).

Fig. 3.. Meis1, Menin and MLL1 are essential for maintaining self-renewal program.

(A) CFU serial replating assay of mouse MIG-CRENpm1c/Dnmt3a mutant cell line (SIIIL12) with dimethyl sulfoxide (DMSO) or VTP-50469. Data represent the mean of three independent experiments. d7, day 7. (B) CFU assay of mouse SIIIL12 cells transduced with MSCV-PURO control (left) or Meis1-PURO (right) virus and grown in the presence of 10 nM VTP-50469. Data represent the mean of three independent experiments. (C) CFU assay of SIIIL12 cells electroporated with control or Meis1- or Rpa3-targeting single guide RNAs (sgRNAs) for Cas9-mediated KO. Data represent the mean of three independent experiments. (D) Chromatin immuno- precipitation sequencing (ChIP-seq) density plots showing changes in chromatin occupancy of Menin, MLL, and H3K4me3 and changes in mRNA expression in response to VTP-50469 in OCI-AML3 cells at the MEIS1 and HOXA loci. (E and F) MEIS1 (E) and HOXB5 (F) gene expression in OCI-AML3 cells transduced with control, sgMLL1, sgMLL2, and sgMenin. Data represent the mean of three independent experiments. One-way ANOVA was performed. Error bars indicate mean ± SD. ns, not significant; *P < 0.05; **P < 0.01; ***P <0.001.

Fig. 4.. Preleukemic Npm1c LT-GMPs and human AML cells can be eradicated by Menin inhibition.

(A and B) Percent engraftment of CD45.2 in peripheral blood (A) and Kaplan-Meier survival analysis (B) of mice transplanted with Npm1c/Dnmt3a LT-GMPs receiving control or 0.1% VTP-50469–spiked chow for 9 weeks (one-way ANOVA; n = 3 mice per group; error bars indicate mean ± SD). (C and D) Percent engraftment of hCD45 in peripheral blood (C) and aplan-Meier survival analysis (D) of NPM1c,FLT3ITD,FLT3TKD-transplanted PDX mice receiving control or 0.1% VTP-50469–spiked chow for 129 days (patient 1, table S5; n = 5 mice per group; error bars indicate mean ± SD). (E) Mutational screening of 49 paired MDS and sAML patient samples for RUNX1, TP53, NPM1, FLT3, ASXL1, DNMT3A, IDH1, and IDH2 mutations revealed six patients with persistent NPM1 mutations detected in MDS samples before AML development. IPSS, International Prognostic Scoring System; SNP, single- nucleotide polymorphism; CN-AML, cytogenetically normal AML; CNAs, copy number alterations. ns, not significant; **P < 0.01; ***P < 0.001.