ΔNp73 overexpression promotes resistance to apoptosis but does not cooperate with PML/RARA in the induction of an APL-leukemic phenotype

Abstract

Here, we evaluated whether the overexpression of transcriptionally inactive ΔNp73 cooperates with PML/RARA fusion protein in the induction of an APL-leukemic phenotype, as well as its role in vitro in proliferation, myeloid differentiation, and drug-induced apoptosis. Using lentiviral gene transfer, we showed in vitro that ΔNp73 overexpression resulted in increased proliferation in murine bone marrow (BM) cells from hCG-PML/RARA transgenic mice and their wild-type (WT) counterpart, with no accumulation of cells at G2/M or S phases; instead, ΔNp73-expressing cells had a lower rate of induced apoptosis. Next, we evaluated the effect of ΔNp73 on stem-cell self-renewal and myeloid differentiation. Primary BM cells lentivirally infected with human ΔNp73 were not immortalized in culture and did not present significant changes in the percentage of CD11b. Finally, we assessed the impact of ΔNp73 on leukemogenesis or its possible cooperation with PML/RARA fusion protein in the induction of an APL-leukemic phenotype. After 120 days of follow-up, all transplanted mice were clinically healthy and, no evidence of leukemia/myelodysplasia was apparent. Taken together, our data suggest that ΔNp73 had no leukemic transformation capacity by itself and apparently did not cooperate with the PML/RARA fusion protein to induce a leukemic phenotype in a murine BM transplantation model. In addition, the forced expression of ΔNp73 in murine BM progenitors did not alter the ATRA-induced differentiation rate in vitro or induce aberrant cell proliferation, but exerted an important role in cell survival, providing resistance to drug-induced apoptosis.

MLL5 improves ATRA driven differentiation and promotes xenotransplant engraftment in acute promyelocytic leukemia model

“…Recombinant lentivirus encoding the PML-RARA fusion transcript (bcr1 isoform) and the Hs MLL5α gene (hereinafter called MLL5 gene) were generated using pCDH1-MCS1-EF1-GFP-Puro (pMEG) lentivector (#CD713B-1; System Biosciences, USA) in HEK293T cells according to the three-plasmid packaging procedure as described elsewhere 11 . The complete optimized sequence of MLL5 gene was synthesized and fully sequenced in both directions by GenScript (Piscataway Township, USA).…”

Reduced SLIT2 is Associated with Increased Cell Proliferation and Arsenic Trioxide Resistance in Acute Promyelocytic Leukemia

“…Recombinant lentivirus encoding the short hairpin sequence (shRNA) targeting the human and murine SLIT2 gene (TRCN0000363636 (human) and TRCN 0000328111 (mouse)) (hereafter called shSLIT2 and shSlit2 for the human and mouse constructs, respectively) were generated using a MISSION pLKO.1 lentivector (Sigma-Aldrich) in HEK293T cells according to the three-plasmid packaging procedure described elsewhere [36]. The MISSION-shRNA sequence selection was performed using the Broad Institute RNAi consortium data bank [37].…”

“…For APL phenotype induction, 1 × 10 6 transduced leukemic blast cells from hCG-PML-RARA mice (expressing the CD45.2) [36] were transplanted into lethally irradiated eight-week-old Pep boy mice (B6.SJL-Ptprca Pepcb/BoyJ, The Jackson Laboratory). After 2 weeks, chimerism was evaluated by CD45.1 (Pep boy, provided by Jackson Laboratory -B6.SJL-Ptprca Pepcb/BoyJ) and CD45.2 markers (Becton-Dickinson) by flow cytometry in peripheral blood.…”