U2af1 is a haplo-essential gene required for hematopoietic cancer cell survival in mice

Abstract

Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained WT allele is expressed, suggesting that mutant hematopoietic cells may require the residual WT allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knockout mice were similar to those in control mice, but that deletion of the WT allele in U2AF1(S34F) heterozygous mutant-expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of WT U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1(S34F)-expressing cells were also more sensitive to reduced expression of WT U2AF1 than nonmutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the WT U2af1 allele was deleted compared with when it was not deleted. These results suggest that selectively targeting the WT U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.

Keywords: Cancer; Hematology; Leukemias; Oncology; RNA processing.

Figure 1. Generation of a conditional U2af1-KO allele.

(A) Targeting strategy to insert loxP sites flanking U2af1 exon 2. (B) Successful targeting of 5 ES cell clones was verified by Southern blotting after digestion with the restriction enzyme AvrII. WT C57BL/6 (B6) DNA was used as a control, and clone 243 was used to generate the U2af1-KO mouse. Neo, neomycin resistance gene; LA, long arm; MA, middle arm; SA, short arm.

Figure 2. Embryonic deletion of U2af1 in hematopoietic cells reduces the number of myeloid, lymphoid, and hematopoietic stem and progenitor cells.

(A) Comparison of fetal liver development of embryos at E14.5 between Vav1-Cre U2af1^WT/fl and Vav1-Cre U2af1^fl/fl mice; arrowheads indicate fetal liver. (B) E14.5 fetal liver hematopoietic cellularity and absolute counts of neutrophils, monocytes, and B cells in the fetal liver of the Vav1-Cre, U2af1^fl/fl, and Vav1-Cre U2af1^WT/fl embryos were compared with those of Vav1-Cre U2af1^fl/fl embryos (n = 3–10). (C) Proportion of progenitor cells (KL and KLS) of Vav1-Cre U2af1^WT/fl compared with Vav1-Cre U2af1^fl/fl mice. (D) Absolute numbers of hematopoietic stem and progenitor cells (KLS, MPP, ST-HSC, LT-HSC, SLAM) in the fetal liver of Vav1-Cre, U2af1^fl/fl, and Vav1-Cre U2af1^WT/fl embryos compared with Vav1-Cre U2af1^fl/fl embryos (n = 3–10). (E) Colony numbers from 25,000 bulk fetal liver cells isolated from Vav1-Cre, U2af1^fl/fl, and Vav1-Cre U2af1^WT/fl embryos compared with Vav1-Cre U2af1^fl/fl embryos after 8 days (n = 4–7). All data are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Tukey’s multiple-comparison test.

Figure 3. U2af1 deletion in adult mice induces multilineage bone marrow failure.

(A) Experimental design of a noncompetitive transplant of whole bone marrow cells from CD45.2 Mx1-Cre, U2af1^fl/fl, Mx1-Cre U2af1^WT/fl, or Mx1-Cre U2af1^fl/fl mice transplanted into lethally irradiated congenic WT CD45.1 recipient mice, followed by pIpC-induced Cre activation and U2af1 deletion. Analysis of the peripheral blood and bone marrow was done 8 to 11 days after pIpC. (B) Absolute numbers of peripheral WBCs, platelets (PLT), RBCs, neutrophils, monocytes, B cells, and T cells of mice transplanted with whole bone marrow from Mx1-Cre, U2af1^fl/fl, and Mx1-Cre U2af1^WT/fl mice compared with Mx1-Cre U2af1^fl/fl mice 8–11 days after pIpC. (C) Bone marrow cellularity of Mx1-Cre, U2af1^fl/fl, and Mx1-Cre U2af1^WT/fl mice compared with Mx1-Cre U2af1^fl/fl mice 8–11 days after pIpC. (D) Total number of neutrophils, monocytes, B cells, and T cells per tibia at 8 to 11 days after pIpC for Mx1-Cre, U2af1^fl/fl, and Mx1-Cre U2af1^WT/fl mice compared with Mx1-Cre U2af1^fl/fl mice. (E) Number of progenitor cells (KL and KLS) per tibia of pIpC-treated mice transplanted with Mx1-Cre, U2af1^fl/fl, and Mx1-Cre U2af1^WT/fl bone marrow cells compared with Mx1-Cre U2af1^fl/fl bone marrow cells 8–11 days after pIpC. (F) H&E staining of bone marrow from Cre-ERT2 U2af1^fl/fl and Cre-ERT2 control mice at 3 days after tamoxifen. The experimental design is similar to that in panel A, with tamoxifen used for induction of U2af1 deletion. All data are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Tukey’s multiple-comparison test. n = 5 per genotype. Scale bars: 100 μm.

Figure 4. Hematopoietic stem cells are dependent on U2af1 expression for survival.

(A) Experimental design of a competitive transplant of whole bone marrow cells from CD45.2 Mx1-Cre, U2af1^fl/fl, Mx1-Cre U2af1^WT/fl, or Mx1-Cre U2af1^fl/fl mice mixed at a 1:1 ratio with congenic WT CD45.1/45.2 competitor cells followed by transplantation into lethally irradiated congenic WT CD45.1 recipient mice followed by pIpC-induced U2af1 deletion. Peripheral blood and bone marrow chimerism of Mx1-Cre, U2af1^fl/fl, and Mx1-Cre U2af1^WT/fl mice was compared with that of Mx1-Cre U2af1^fl/fl mice. (B) Bulk peripheral blood chimerism (n = 9–10). (C and D) Myeloid peripheral blood cell chimerism (n = 9–10). (E and F) Lymphoid peripheral blood cell chimerism (n = 9–10). (G) Bone marrow chimerism of mature hematopoietic cells (neutrophils, monocytes, B cells, and T cells) at 10 months after pIpC (n = 8). (H) Bone marrow chimerism of stem and progenitor cells (KL, KLS, MPP, ST-HSC, LT-HSC, SLAM) 10 months after pIpC (n = 8 for KL and KLS; n = 3–4 for all other). All data are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, 2-way (B–F) or 1-way (G and H) ANOVA, with Tukey’s multiple-comparison test.

Figure 5. U2af1 deletion induces alternative splicing, but there is no change or alteration in the sequence specificity at the 3′ splice site.

Differential splicing analysis of RNA-Seq of E14.5 hematopoietic progenitor cells (KL). (A) Distribution of alternative splicing events in Vav1-Cre U2af1^fl/fl compared with Vav1-Cre control cells (left), Vav1-Cre U2af1^WT/fl compared with Vav1-Cre control cells (center), and Vav1-Cre U2af1^WT/S34F compared with Vav1-Cre control cells (right). (B) Venn diagram comparing the skipped exon events (positive and negative) in Vav1-Cre U2af1^fl/fl, Vav1-Cre U2af1^WT/fl, and Vav1-Cre U2af1^WT/S34F. (C–E) The consensus sequence around the 3′ AG dinucleotide splice-acceptor site of cassette exons that are unchanged (control), included more (positive), or skipped more (negative) in Vav1-Cre U2af1^fl/fl, Vav1-Cre U2af1^WT/fl, and Vav1-Cre U2af1^WT/S34F compared with Vav1-Cre control (FDR < 0.1, |ΔΨ| > 0.1). n = 1–4 per genotype. A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice site; MXE, mutually exclusive exons; RI, retained intron; negative, decrease in event relative to Vav1-Cre control; positive, increase in event relative to Vav1-Cre control.

Figure 6. Survival of mutant U2AF1(S34F) hematopoietic cells is dependent on the expression of the residual WT allele and the ratio of U2AF1(WT:S34F) expression.

(A) Experimental design of a competitive transplant of whole bone marrow cells from CD45.2 Mx1-Cre, Mx1-Cre U2af1^WT/fl, Mx1-Cre U2af1^fl/fl, Mx1-Cre U2af1^WT/S34F, or Mx1-Cre U2af1^fl/S34F mice mixed at a 1:1 ratio with congenic WT CD45.1/45.2 competitor cells followed by transplantation into lethally irradiated congenic WT CD45.1 recipient mice. U2af1 deletion was induced by pIpC, and analysis of the peripheral blood chimerism was performed. (B) Peripheral blood neutrophil chimerism of Mx1-Cre, Mx1-Cre U2af1^WT/fl, Mx1-Cre U2af1^fl/fl, Mx1-Cre U2af1^WT/S34F, and Mx1-Cre U2af1^fl/S34F mice (n = 14–16). (C) Experimental design of a competitive transplant of whole bone marrow cells from CD45.2 tgU2AF1(WT)/U2af1^WT/WT, tgU2AF1(WT)/U2af1^fl/fl, tgU2AF1(S34F)/U2af1^WT/WT, or tgU2AF1(S34F)/U2af1^fl/fl mice. All the donor test mice have both Mx1-Cre and rtTA. Transgenic U2AF1(WT) and U2AF1(S34F) were induced by 10,000 ppm doxycycline (dox) chow, followed by pIpC-induced U2af1 deletion after 4 weeks, and analysis of the peripheral blood chimerism was performed. (D) Overall peripheral blood and neutrophil chimerism in tgU2AF1(WT)/U2af1^WT/WT, tgU2AF1(WT)/U2af1^fl/fl, tgU2AF1(S34F)/U2af1^WT/WT, and tgU2AF1(S34F)/U2af1^fl/fl mice after induction of transgenic U2AF1(WT) or U2AF1(S34F) by 10,000 ppm doxycycline chow and U2af1 deletion induced by pIpC Cre-activation (n = 7–8). (E) Experimental design of a competitive transplant of whole bone marrow cells from CD45.2 Mx1-Cre/rtTA/tgU2AF1(WT)/U2af1^WT/S34F (tgU2AF1[WT]/U2af1^WT/S34F) mice. Transgenic U2AF1(WT) was induced by 10,000 ppm doxycycline chow starting 2 weeks after transplant, followed by pIpC-induced U2af1 S34F mutant allele activation after 2 weeks of doxycycline chow. Analysis of peripheral blood chimerism was performed. (F) Overall peripheral blood and neutrophil chimerism of tgU2AF1(WT)/U2af1^WT/S34F with or without doxycycline chow treatment (n = 9–10). All data are represented as mean ± SD. *P < 0.05; ***P < 0.001, 2-way ANOVA with Šidák’s (B and F) or Tukey’s (D) multiple-comparison test.

Figure 7. Hematopoietic stem cells expressing mutant U2AF1(S34F) are more sensitive to decreased levels of the WT U2AF1 expression than WT cells.

All the donor test mice have Mx1-Cre and rtTA. (A) Experimental competitive transplant design of test whole bone marrow cells from CD45.2 tgU2AF1(WT)/U2af1^fl/fl or tgU2AF1(WT)/U2af1^fl/S34F mice mixed at a 1:1 ratio with congenic WT CD45.1/45.2 competitor cells, followed by transplantation into lethally irradiated congenic WT CD45.1 recipient mice. Transgenic U2AF1(WT) expression was induced by 10,000 ppm doxycycline chow starting 2 weeks after transplant followed by pIpC induction of U2af1 deletion and S34F mutant allele activation 2 weeks later. Two weeks after pIpC, the doxycycline dose was reduced to 2500 ppm or 1250 ppm. (B) Peripheral blood neutrophil chimerism of tgU2AF1(WT)/U2af1^fl/fl and tgU2AF1(WT)/U2af1^fl/S34F after induction of transgenic U2AF1(WT) by 2500 ppm doxycycline chow (n = 5). (C) Peripheral blood neutrophil chimerism of tgU2AF1(WT) U2af1^fl/fl and tgU2AF1(WT)/U2af1^fl/S34F after induction of transgenic U2AF1(WT) by 1250 ppm doxycycline chow (n = 5). (D) Experimental noncompetitive transplant design of whole bone marrow cells from CD45.2 tgU2AF1(WT)/U2af1^fl/S34F mice transplanted into lethally irradiated congenic WT CD45.1 recipient mice for KL cell RNA-Seq. (E) Distribution of alternative splicing events in tgU2AF1(WT)/U2af1^fl/S34F cells after Mx1-Cre induction by pIpC and transgenic U2AF1(WT) induction by 10,000 ppm, 2500 ppm, or 1250 ppm doxycycline chow, compared with the untreated tgU2AF1(WT)/U2af1^fl/fl control. (F and G) ΔΨ of tgU2AF1(WT)/U2af1^fl/S34F cells after Mx1-Cre induction and transgenic U2AF1(WT) induction by 10,000 ppm, 2500 ppm, or 1250 ppm doxycycline chow compared with the untreated tgU2AF1(WT)/U2af1^fl/fl control cells (n = 3–6, FDR < 0.1, |ΔΨ| > 0.1). All data are represented as mean ± SD. Box plots show mean ± minimum and maximum. *P < 0.05; ***P < 0.001, 2-way (B and C) or 1-way (F and G) ANOVA with Tukey’s multiple-comparison test.

Figure 8. Hematopoietic cancer cells expressing mutant U2AF1(S34F) are sensitive to decreased levels of WT U2AF1 expression.

(A) Experimental design of transplantation of tgU2AF1(WT)/U2af1^fl/S34F MLL-AF9 AML tumor cells (GFP⁺CD45.2⁺) isolated from the spleen of primary mice into sublethally irradiated secondary recipients. Secondary recipients were treated with or without 10,000 ppm doxycycline chow followed by pIpC induction and analysis of the peripheral blood and tumor watch. (B) GFP⁺ MLL-AF9 AML cell chimerism up to 21 days after the second pIpC dose (n = 10). (C) Kaplan-Meier survival curves up to 93 days after transplant (n = 10). All data are represented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Tukey’s multiple-comparison test.