Rara haploinsufficiency modestly influences the phenotype of acute promyelocytic leukemia in mice

Abstract

RARA (retinoic acid receptor alpha) haploinsufficiency is an invariable consequence of t(15;17)(q22;q21) translocations in acute promyelocytic leukemia (APL). Retinoids and RARA activity have been implicated in hematopoietic self-renewal and neutrophil maturation. We and others therefore predicted that RARA haploinsufficiency would contribute to APL pathogenesis. To test this hypothesis, we crossed Rara(+/-) mice with mice expressing PML (promyelocytic leukemia)-RARA from the cathepsin G locus (mCG-PR). We found that Rara haploinsufficiency cooperated with PML-RARA, but only modestly influenced the preleukemic and leukemic phenotype. Bone marrow from mCG-PR(+/-) × Rara(+/-) mice had decreased numbers of mature myeloid cells, increased ex vivo myeloid cell proliferation, and increased competitive advantage after transplantation. Rara haploinsufficiency did not alter mCG-PR-dependent leukemic latency or penetrance, but did influence the distribution of leukemic cells; leukemia in mCG-PR(+/-) × Rara(+/-) mice presented more commonly with low to normal white blood cell counts and with myeloid infiltration of lymph nodes. APL cells from these mice were responsive to all-trans retinoic acid and had virtually no differences in expression profiling compared with tumors arising in mCG-PR(+/-) × Rara(+/+) mice. These data show that Rara haploinsufficiency (like Pml haploinsufficiency and RARA-PML) can cooperate with PML-RARA to influence the pathogenesis of APL in mice, but that PML-RARA is the t(15;17) disease-initiating mutation.

Figure 1

Preleukemic cooperation of Rara haploinsufficiency and PML-RARA. Immunophenotype of bone marrow cells from the indicated littermate-matched mice at 8 weeks. (A-B) Myeloid immunophenotype by Gr1, CD115, cKit, and CD34. (C-D) Progenitor immunophenotype by lineage (CD3, CD8, CD4, CD19, B220, Gr1, and Terr-119), cKit, Sca, CD34, and FcγRIII-R. (E) Peripheral blood immunophenotype by CD3 and Gr1. (F) Neutrophil counts assessed by Coulter counter at 4 and 6 months of age.

Figure 2

Ex vivo growth of myeloid bone marrow cells. (A) Experimental schema. (B) Colony formation and serial replating. Bone marrow cells from littermate-matched mice at 8 weeks of age were plated in duplicate in methylcellulose containing IL-3, IL-6, and SCF, and after 7 days, colonies and total cells per duplicate plates were counted. Cells were then replated in duplicate and 7 days later, colonies were counted and replated. Replating continued for 4 weeks or until colony formation failed. (C) Methylcellulose colony growth. On day 7 of week 1, total colonies and total cells on duplicate were counted to assess the average number of cells per colony. (D) Immunophenotype of day-7, week-1 total cells from duplicate methylcellulose plates.

Figure 3

Competitive repopulation. (A) Experimental schema. (B) Bone marrow cells from the indicated mice at 8 weeks of age were mixed at a ratio of 1 to 9 with competitor CD45.1 bone marrow cells from sex- and age-matched mice. These cells were transplanted into sex-matched, 6-week-old, lethally irradiated CD45.1 recipients. At the indicated time points, peripheral blood was assessed for ratios of CD45.2⁺ and CD45.1⁺ WBCs. (C) At 6 months of age, peripheral blood from mCG-PR^+/− × Rara^+/− recipients was assessed for ratios of CD45.2⁺ vs CD45.1⁺ cells in Gr1⁺, CD19⁺, and CD3⁺ compartments.

Figure 4

Tumor watch. (A) The indicated cohorts of littermate-matched mice were prospectively established in a tumor watch. Mice were followed for 18 months and moribund animals were killed. Leukemia occurred in mCG-PR^+/− mice, but not in littermate controls. There was no significant difference in latency or penetrance in mCG-PR^+/− × Rara^+/− vs mCG-PR^+/− × Rara^+/+ mice. (B) Maximum diameter of largest cervical lymph node at the time of killing. (C) Spleen size in the indicated mice at the time of killing or at 18 months of age, whichever came first. (D) Peripheral WBC counts at the time of killing or at 18 months of age. (E) Mice shown in Figure 3 were followed, and moribund mice were killed. There was no significant difference in survival by genotype of transplanted bone marrow cells.

Figure 5

Analysis of mCG-PR^+/− × Rara^+/− and mCG-PR^+/− × Rara^+/+ leukemia cells. (A) Expression array profiling of leukemic spleen cells from 5 mCG-PR^+/− × Rara^+/− and 5 mCG-PR^+/− × Rara^+/+ mice using Affymetrix Exon 1.0 arrays. Tumors did not cluster by genotype using an unsupervised analysis. (B-D) Ex vivo ATRA sensitivity. (B) Experimental schema. (C) Cryopreserved spleen cells from 9 mCG-PR^+/− × Rara^+/− and 9 mCG-PR^+/− × Rara^+/+ independent tumors were thawed and cultured for 48 hours in liquid medium containing IL-3, IL-6, and SCF ± 1μM ATRA. Viable cells were counted and plated in methylcellulose containing IL-3, IL-6, and SCF. After 7 days, colonies (C) were counted and pairwise assessed for ATRA sensitivity (D).