Molecular pathogenesis of disease progression in MLL-rearranged AML

Abstract

Leukemic relapse is frequently accompanied by progressively aggressive clinical course. To understand the molecular mechanism of leukemic relapse, MLL/AF9-transformed mouse leukemia cells were serially transplanted in C57BL/6 mice (N = 96) by mimicking repeated recurrences, where mutations were monitored by exome sequencing (N = 42). The onset of leukemia was progressively promoted with advanced transplants, during which increasing numbers of somatic mutations were acquired (P < 0.005). Among these, mutations in Ptpn11 (p.G60R) and Braf (p.V637E) corresponded to those identified in human MLL-AML, while recurrent mutations affecting Msn (p.R295C) were observed only in mouse but not in human MLL-AML. Another mutated gene of interest was Gnb2 which was reported to be recurrently mutated in various hematological neoplasms. Gnb2 mutations (p.G77R) were significantly increased in clone size (P = 0.007) and associated with earlier leukemia onset (P = 0.011). GNB2 transcripts were significantly upregulated in human MLL-AML compared to MLL-negative AML (P < 0.05), which was supported by significantly increased Gnb2 transcript induced by MLL/AF9 overexpression (P < 0.001). In in vivo model, both mutation and overexpression of GNB2 caused leukemogenesis, and downregulation of GNB2 expression reduced proliferative potential and survival benefit, suggesting a driver role of GNB2. In conclusion, alterations of driver genes over time may play an important role in the progression of MLL-AML.

Fig. 1

Serial transplantation of mouse MLL/AF9-acute myeloid leukemia (AML) (N = 96). a Serial transplantation of MLL/AF9-transduced granulocyte-monocyte progenitor (GMP) cells in C57BL/6 mice. b Total white blood cell (WBC) counts and c GFP-positive WBC counts on day 14. Numbers of mutations identified in strain A1 (d) and B1 (e)

Fig. 2

Whole exome sequencing (WES) of mouse MLL/AF9-AML. a, b Upper panels showed 12 and 13 mice analyzed by WES in strain A1 and B1, respectively. Middle panels demonstrated landscape of somatic mutations in each strain. Green and gray indicated shared and private mutations, respectively. Driver mutations were represented in bold italic font. In lower panels, fishplots displayed clonal architecture in A1-4-7 and B1-4-5 mice according to the results of mutational landscape in their own and their ancestry transplant generations. c–e Upper panels showed each 3 mice per strain from strain C1, C2, and E1, respectively. Lower panels demonstrated landscape of somatic mutations in each strain

Fig. 3

Mutational spectrum in human MLL-AML (N = 168). A frequency of mutations (%) was shown in each affected leukemogenic pathway

Fig. 4

Impact of driver mutations on leukemia onset and clonal expansion in MLL/AF9-AML. a, b Comparison of leukemia onset time between cases with and without driver mutations (a) or Gnb2 mutations (b). c, d Assessment of trend in variant allele frequencies of Msn (c) or Gnb2 (d) mutations

Fig. 5

GNB2 expression in human MLL-AML. a, b GNB2 relative expression in human AML with and without MLL-fusion gene (a RNA sequencing and b expression array). c GNB2 relative expression in bone marrow mononuclear cells (BMMNCs) from healthy donors (N = 5), and AML cell lines with (N = 3) and without MLL-fusion gene (N = 4) (real-time RT-PCR). Each experiment was performed in triplicate. The Benjamini–Hochberg procedure was applied to the correction of multiple testing. d Gnb1 and Gnb2 expression levels were assessed in comparison between empty- and MLL/AF9-transduced Ba/F3 cells. Each experiment was performed in triplicate

Fig. 6

Cytokine independency due to GNB2. a Growth curves of Ba/F3 cells cultured without IL-3 after transduction of wild type or mutated (p.G77R) GNB2. Each experiment was performed in triplicate. b Protein levels of total Akt and phosphorylated Akt (p-Akt) were evaluated by immunoblot

Fig. 7

Oncogenic potential of mutant and wild-type GNB2. Ba/F3 cells transduced with wild-type GNB2, GNB2 mutant, and mock were transplanted in immunodeficient mice. a, b, c To study GNB2-associated tumorigenesis, 1 × 10⁷ cells per experiment were subcutaneously injected into nude mice (N = 30 in total). The tumor size was measured (a on day 23 and b on day 42) and compared between 3 groups (c). d, e, f To assess GNB2-associated leukemic involvement, 2 × 10⁶ cells per mouse were intravenously transplanted into NOD/SCID/γc null (NOG) mice (N = 21 in total). GFP positivity was measured in bone marrow cells (d on day 15 and e on day 28). Survival time was compared between 3 groups (f)

Fig. 8

Impact of decreased GNB2 expression or depleted Gnb2 genome in MLL-AML. a, b, c Growth curves (cell numbers) and GNB2 expression values of 3 human MLL-AML cell lines (a NOMO1, b MOLM13, and c THP1) treated with GNB2 shRNA and control scramble shRNA. Each experiment was performed in triplicate. d, e By genome-wide CRISPR-Cas9 screens, read counts of six sgRNAs targeting per exon (exon 1 (d) and 2 (e)) in Gnb2 were measured after a 16-day incubation period to confirm potential oncogenic function of Gnb2 in MLL/AF9-AML