The Effect of Rara Haploinsufficiency in a Mouse Model of Acute Promyelocytic Leukemia.
Authors: Welch John S. , Ley Timothy 
Published: October 2019

Abstract 3475
Poster Board III-412
Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation, which leads to expression of the fusion protein that initiates this disease (PML-RARA), the creation of a small and inconsistently expressed RARA-PML fusion protein, and haploinsufficiency for both RARA and PML. While alternative translocations and fusion proteins have been associated with APL, RARA appears to be a necessary member of each (e.g. PLZF-RARA, NPM-RARA, STAT5b-RARA, NuMA-RARA). Furthermore, RARA activity is important for myeloid maturation. We have therefore explored the effect of RARA haploinsufficiency in the murine Cathepsin-G PML-RARA (mCG-PR) model of APL.
We crossed RARA+/− mice with mCG-PR mice, all on a C57/B6 background. Both mCG-PR and the mutant RARA allele were observed at normal Mendelian ratios. At 8 weeks, mCG-PR x RARA+/− mice exhibited normal peripheral blood counts, spleen sizes and total bone marrow cells. Bone marrow cells from 8-week old mCG-PR x RARA+/− and mCG-PR mice both exhibited increased self-renewal in methylcellulose replating assays, with increased average cells per colony in mCG-PR x RARA+/− and mCG-PR colonies compared to wild type (28,470 ± 6,000 and 15,400 ± 2,375 vs 7,700 ± 630, p = 0.0001 and 0.0003 following the initial plating). Five months after bone marrow transplantation at a 1:9 ratio with competitor Ly5.1 bone marrow cells, mCG-PR derived cells have engrafted and expanded in four recipients to 10%, 12.4%, 13.8% and 15.2% of peripheral blood cells, while mCG-PR x RARA+/− derived cells have more robustly expanded to 8.7%, 20.2%, 24.5%, and 30.3% of peripheral blood cells.
A cohort of 29 mCG-PR x RARA+/− and 20 mCG-PR mice was subjected to a tumor watch. With an average follow up of 10 months, we have observed AML arise in 9 mCG-PR x RARA+/− mice and 11 mCG-PR mice (an additional 20 and 9 mice in each respective cohort remain at risk of leukemia and will be subsequently evaluated). Leukemia arising from mCG-PR mice exhibited leukocytosis, splenomegaly and variable myeloid differentiation arrest in the peripheral blood and spleen, as measured by manual differential counts and cKit/CD11b expression in the Gr1+ compartment. Leukemia from two mice had marked promyelocytic maturation arrest, but 5 others retained differentiation, with 30-60% bands and ring-formed neutrophils in the spleen, which correlated with similar percentages of Gr1+/cKit-/CD11b+ cells (4 mice died with splenomegaly noted during necropsy and were not further characterized). AML arising in mCG-PR x RARA+/− mice displayed similar variability in maturation arrest, but these mice had a trend towards lower peripheral blood WBC counts compared to mCG-PR AML (mean WBC 28,000/mcl ± 26,000 vs 82,000/mcl ± 71,000/mcl, p = 0.06) and larger spleen size (mean 1,425 mg ± 475 mg vs 1,017 mg ± 193 mg, p = 0.03). In addition, 6/9 mCG-PR x RARA+/− mice with AML had marked cervical lymphadenopathy caused by infiltrating AML cells. Only 1/11 mCG-PR mice with AML displayed lymphadenopathy, which previously has been observed only rarely with this mouse model (Westervelt et al, Blood 2003). Both mCG-PR x RARA+/− and mCG-PR AML cells responded to ATRA with increased maturation in vitro and in vivo, a shift from cKit+/CD11b- to cKit-/CD11b+ expression, a loss of self renewal capacity, and improved survival compared to untreated controls, as expected.
In sum, mCG-PR x RARA+/− mice are different from mice with mCG-PR alone. RARA haploinsufficiency further perturbs hematopoiesis in mCG-PR mice, accelerating an early competitive advantage conferred by PR expression, and possibly altering the pattern of homing and peripheralization of AML cells. Lower peripheral white blood cell counts in these animals more accurately reproduce the relatively low white counts of most patients with APL. These data strongly suggest that RARA haploinsufficiency contributes to the overall phenotype of APL initiated by PML-RARA, and thus is a relevant mutation created by the t(15;17) translocation.

Disclosures:
Welch: Cephalon: Research Funding; Eisai: Research Funding.