Abstract 1011: NOTCH1 signaling is essential for leukemia initiating cell self-renewal in T-ALL
Authors: Ma Wenxue , Gutierrez Alejandro , Wei Ping , Sadarangani Anil , Goff Daniel J. , Shih Alice Y. , Court Angela C. , Jiang Qingfei , Leu Heather , Wall Russell H. , Crews Leslie A. , Look A. Thomas , Jamieson Catriona H. M. 
Published: August 2012

Abstract
Leukemia initiating cells (LIC) contribute to therapeutic resistance through mutations in cellular self-renewal and survival pathways. NOTCH1 mutations are common in T-cell acute lymphoblastic leukemia (T-ALL). However, the role of NOTCH1 activation in human LIC propagation has not been established. Pediatric T-ALL serially transplantable LIC were found to be enriched in the CD34+CD4− and CD34+CD7− fractions of newly diagnosed patient samples. More recently, a CD7+CD1a− glucocorticoid resistant LIC population, capable of engrafting leukemia in NOD/SCID IL2R gamma null (NSG) mice, was identified in primary adult T-ALL. To identify the molecularly characterized potential LIC populations in pediatric T-ALL without proceeding in vitro culture and examine the role of NOTCH1 activation in LIC propagation. 12 pediatric T-ALL samples were sequenced for NOTCH1 mutation examination. Humanized LIC mouse models were established and dosed with either NOTCH1 mAb or IgG1 mAb control at 10 mg/kg intraperitoneally every 4 days for 6 doses. Mice were sacrificed one day after the last dose, and hematopoietic organs were collected for FACS analysis. To further define the LIC populations in pediatric T-ALL, CD34+CD38+CD2+CD7+Lin− and CD34+CD38+CD2+CD7−Lin− cells were isolated from T-ALL primary patients’ blood by FACS sorting and transplanted into neonatal RAG2−/−γc−/− mice to determine their leukemic engraftment potential. Serial transplantations were done for testing the LIC self-renewal capacity. Mouse hematopoietic organs were collected for FACS analysis, mouse brains were sectioned for human cells examination by immunohistochemistry. NOTCH1 and its downstream gene expressions were examined by q-RT-PCR between the T-ALL CD34+ and CD34− populations. Six of 12 pediatric T-ALL patient samples were found NOTCH1 mutation. Mice transplanted with CD34+ and CD34+CD2+CD7+ or CD34+CD2+CD7− cells developed a T-ALL-like disease characterized by pale BM and enlarged spleen, thymus and liver. Human CD34+ enriched cells from NOTCH1 mutated T-ALL maintained leukemic engraftment while an equivalent number of CD34+ cells from NOTCH1 wild type T-ALL did not. T-ALL CD34+ progenitors from NOTCH1 mutated T-ALL have a significant higher engraftment in BM when compared with those from NOTCH1 wild type T-ALL. CD34+CD2+CD7+ and CD34+CD2+CD7− populations are more prominent in NOTCH1 mutated samples. Both the human CD34+ and CD34+CD2+CD7+ populations were significantly reduced in BM when treated with hN1 mAb in vivo. NOTCH1 and its downstream genes expression were significantly reduced in NOTCH1 mutated CD34+ cells when compared with CD34− cells. Human T-ALL LIC have enhanced NOTCH1 expression; CD34+CD2+CD7+ and CD34+CD2+CD7− subpopulations are enriched for LIC activity in pediatric T-ALL; A selective hN1 mAb inhibits human T-ALL LIC survival and self-renewal in vivo.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1011. doi:1538-7445.AM2012-1011