A somatic mutation in moesin drives progression into acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) arises when leukemia-initiating cells, defined by a primary genetic lesion, acquire subsequent molecular changes whose cumulative effects bypass tumor suppression. The changes that underlie AML pathogenesis not only provide insights into the biology of transformation but also reveal novel therapeutic opportunities. However, backtracking these events in transformed human AML samples is challenging, if at all possible. Here, we approached this question using a murine in vivo model with an MLL-ENL fusion protein as a primary molecular event. Upon clonal transformation, we identified and extensively verified a recurrent codon-changing mutation (Arg²⁹⁵Cys) in the ERM protein moesin that markedly accelerated leukemogenesis. Human cancer-associated moesin mutations at the conserved arginine-295 residue similarly enhanced MLL-ENL-driven leukemogenesis. Mechanistically, the mutation interrupted the stability of moesin and conferred a neomorphic activity to the protein, which converged on enhanced extracellular signal-regulated kinase activity. Thereby, our studies demonstrate a critical role of ERM proteins in AML, with implications also for human cancer.

Fig. 1.. Disease characteristics of the iMLL-ENL model.

(A) pGMs were isolated from iMLL-ENL mice and transplanted competitively into lethally irradiated recipients (n = 10). Colored symbols and connecting lines show levels of donor-derived myeloid in individual recipients over time. (B) Left: Strategy used to evaluate the clonal composition of iMLL-ENL transformed AML using lentiviral barcoding. Right: Sanger sequencing of DNA barcodes after transformation, demonstrating clonality in a majority of individual AMLs.

Fig. 2.. iMLL-ENL cells associate with a recurrent codon-changing point mutation in moesin upon AML transformation.

(A) Experimental strategy to identify secondary mutations that associate with transformation into MLL-ENL–driven AML. (B) Predicted deleterious mutations identified in AML samples originating from different LIC subsets. (#1) Three different missense variants were detected in Cdh11: p.G86V, p.I100F, and p.V147D. AA, amino acid. (C) The presence of the MSN R295C mutation in AML samples derived from pGM cells from three separate founders. (#2) Two different missense variants were detected in Ptpn11: p.G60V and p.E76K. *The founder mouse in pGM_4 was found to harbor a low AF of the MSN R295C mutation. (D) The presence of the MSN R295C mutation in AML samples derived from a non–transplantation-based model system. In (B) to (D), each column represents an individual leukemic sample, with dark gray boxes indicating a gain of mutation with an allele frequency (AF) > 0.3 and light gray boxes indicating a gain of mutation with an AF of 0.1 to 0.3. (E) Sanger sequencing chromatograms depicting the Msn missense C>T mutation (leading to the R295C substitution) in leukemic cells and in B and T cells from the same sample. (F) Sanger sequencing of Msn from gDNA and cDNA from two female leukemic samples derived from the same founder mouse, demonstrating that, in Msn mutant cells (experiment pGM_2, mouse 4), Msn expression is derived from the active X chromosome in female cells. The same results were obtained from AML sample mouse 6 in experiment pGM_2, which also presented with the Msn mutation. (G) Ultradeep targeted sequencing results of the Msn C883T substitution in WT C57BL/6 and uninduced iMLL-ENL cells.

Fig. 3.. AML progression is markedly accelerated by C295 mutant MSN and associates with diminished differentiation.

(A) Outline of the experimental strategy used to explore the kinetics of MLL-ENL–driven AML development as a consequence of C295 mutant MSN. (B) Kaplan-Meier survival curves depicting event-free survival of recipients transplanted with iMLL-ENL LICs transduced with either WT (n = 25) or C295 mutant MSN (n = 20). (C) Count of green fluorescent protein–positive (GFP⁺) cells (transduced LICs) in PB of recipients at 14, 21, or 28 days after transplantation (n = 5 mice per group/time point). n.s., not significant. (D) Rapid development of splenomegaly in recipients transplanted with C295 mutant MSN LICs. The image show spleens harvested from recipients 28 days after transplantation. (E) Blast cell frequency at 14, 21, and 28 days after transplantation of iMLL-ENL LICs with WT or C295 mutant MSN (n = 5 mice per group/time point). See also fig. S2. (F) AML with C295 mutant MSN continues to rely on MLL-ENL expression. Event-free survival of secondary recipients of iMLL-ENL and C295 mutant MSN expressing leukemic cells as a consequence of MLL-ENL expression (n = 5 mice per group). (G) Knockdown of C295 mutant MSN restricts the propagation of MLL-ENL–driven AML. Left: MSN expression was measured by Western blot following short hairpin RNA (shRNA)–mediated Msn knockdown. Right: Event-free survival in secondary recipients of MLL-ENL + C295 mutant MSN following shRNA-mediated Msn knockdown (n = 10 per group). A scrambled shRNA was used as control. ACTB, beta-actin.

Fig. 4.. Human cancer-associated MSN mutations accelerate AML progression.

(A) The C295 mutant MSN does not affect normal HSC function or multilineage development. Left: Engraftment of normal candidate HSCs expressing WT or C295 mutant MSN (GFP⁺) in PB at 28 weeks after transplantation. Right: Hematopoietic lineage distribution from WT or C295 mutant MSN (n = 7 to 8 mice per group). (B) MSN is dispensable for disease progression from iMLL-ENL LICs. Kaplan-Meier survival curves depict event-free survival of recipients transplanted with iMLL-ENL LICs transduced with either a scramble shRNA or an shRNA against Msn (n = 10 mice per group). (C) Event-free survival of recipients transplanted with iMLL-ENL LICs transduced with either a phosphoinactive mutant construct (MSN T558A) or a phosphomimetic mutant (MSN T558D) (n = 10 mice per group). (D) Leukemic propagation of C295 mutant MSN AML is impaired by the phosphomimetic T558D mutation. Kaplan-Meier survival curves depict event-free survival of recipients transplanted with iMLL-ENL LICs expressing C295 MSN together with either phosphoinactive T558A (C295 + A558) or phosphomimetic T558D (C295 + D558) variants (n = 10 mice per group). (E) R295 mutant MSN-variants identified in human cancers accelerate MLL-ENL–driven AML progression. Left: MSN variants adjacent to the R295 residue from The Cancer Genome Atlas (TCGA) and Catalog of Somatic Mutations in Cancer databases. UCEC, uterine corpus endometrial carcinoma; GA, gastric adenocarcinoma; MA, melanoma; LC, large intestine carcinoma. Right: Kaplan-Meier survival curves depict event-free survival of recipients transplanted with iMLL-ENL LICs transduced with C293, C294, H295, or S295 MSN variants (n = 5 mice per group). iMLL-ENL LICs cells transduced with WT or C295 MSN (same as in Fig. 3B) are included for reference.

Fig. 5.. The R295C mutation alters the structural dynamics of MSN, while the MSN interactomes is preserved in C295 mutant MSN.

(A) Simulation of molecular dynamics of WT and C295 mutant MSN. Secondary structural changes at the FERM C-terminal domain of mutant MSN are shown in blue. In the C295 mutant structure, the highlighted region (composed of the amino acids Trp²⁴², Ser²⁴³, Glu²⁴⁴, Phe²⁵⁰, and Asn²⁵¹) has transformed from a β sheet and α helix to a random coil structure. (B) Nondenaturing gel electrophoresis of WT and C295 mutant MSN. Western blot with an anti-FLAG antibody was performed using nondenaturing (top) and denaturing (bottom) gel electrophoresis. (C) Densitometry profile showing the mean ratios ± SD of monomeric and dimeric MSN in cells transduced with WT or C295 mutant MSN in three independent experiments. (D) Enriched biological terms associated with MSN interactomes. (E) Volcano plot representation of abundance of interacting proteins of C295 mutant versus WT MSN from five biological replicates. The significance threshold (P < 0.05) is indicated with a dashed line at the y axis and fold change indicated at the x axis.

Fig. 6.. C295 mutant MSN increases ERK phosphorylation in a pMEK-independent manner.

(A) C295 mutant MSN increases ERK phosphorylation but not MEK phosphorylation. Left: Representative immunoblotting analysis showing the levels of pERK, pMEK, total ERK, and total MEK in WT (n = 5), C295 mutant MSN (n = 5), or HRAS L61 (n = 2) transduced LICs. β actin immunoblotting was used as loading control. Right: Summary of the ERK and MEK activity. Graphs show mean pERK/total ERK ± SD and mean pMEK/total MEK ± SD in two to five independent experiments. (B and C) mRNA expression of Myc and Ccnd1 in GFP⁺ MLL-ENL in LICs with WT or C295 mutant MSN at different time points after transplantation. Data show means ± SD from three biological replicates (individual mice) per time point.