Analysis of the Tissue-Specific Expression Requirements and Identification of Cooperating Mutations for Leukemogenesis in an Inducible CALM/AF10 Knock-in Mouse Model
Authors: Dutta Sayantanee , Krause Alexandre , Vosberg Sebastian , Herold Tobias , Ksienzyk Bianca , Quintanilla-Fend Leticia , Tizazu Belay , Graf Alexander , Krebs Stefan , Blum Helmut , Greif Philipp A , Schneider Marlon , Dahlhof Maik , Spiekermann Karsten , Zimber-Strobl Ursula , Wolf Eckhard , Bohlander Stefan K 
Published: October 2019

Abstract
The CALM/AF10 fusion, which is the result of the t(10;11)(p12;q14), is associated with various hematological malignancies including acute myeloid leukemia (AML), T cell acute lymphoblastic leukemia (ALL) and malignant lymphoma, and has usually a poor prognosis. We established a CALM/AF10 knock-in mouse model, which allows tissue-specific expression of the fusion gene. The CALM/AF10 fusion gene, preceded by a loxP site flanked transcriptional stop cassette, was knocked into the Rosa26 locus (R26LSLCA strain). Tissue-specific CALM/AF10 expression was achieved by crossing R26LSLCA mice with three Cre inducer lines expressing the Cre recombinase under the control of defined promoters (Vav-Cre, Mb1-Cre, CD19-Cre). Acute leukemia developed in all (n=23) Vav-Cre/R26LSLCA mice with a median latency of 12 months. In the Vav-Cre line, the Cre recombinase is expressed in all hematopoietic cells including stem cells. Leukemias were either myeloid or had a combination of myeloid and lymphoid features with the expression of the B cell marker B220. The leukemia in these mice was characterized by leukocytosis, splenomegaly and bone marrow as well as multi organ infiltration of myeloid blast like cells. In contrast, none of the mice with the Mb1-Cre/R26LSLCA (n=25) or the CD19-Cre/R26LSLCA (n=20) genotype, which expressed the CALM/AF10 from the early B cell progenitor stage, developed leukemia, even though the B cells of these mice expressed the CALM/AF10 transcript at comparable levels to the levels observed in the bone marrow and spleen cells of the leukemic mice. Affymetrix gene expression profiling (GEP) of leukemic and pre-leukemic bone marrow cells of Vav-Cre/R26LSLCA mice revealed that high expression of Hoxa cluster genes and the Hox co-factor Meis1 occurred before the onset of overt leukemia. The B cells from Mb1-Cre/R26LSLCA mice did not show higher expression of Hoxa cluster genes or of Meis1 compared to B cells from wild type mice. The long latency to leukemia development in the Vav-Cre/R26LSLCA mice suggested that additional genetic lesions were required to cooperate with the CALM/AF10 fusion to lead to malignant transformation. To identify these lesion, we performed whole exome sequencing (WES) on the DNA from the leukemic cells of 8 Vav-Cre/R26LSLCA mice and compared the sequence to the corresponding germ line DNA and a pool of 10 germline control WES datasets. We identified between 1 and 6 somatic point mutations and indels per sample in the 5 exomes with the highest product of percent exome coverage at more than 10x and blast percentage (10x coverage: range 22 to 91%, median 86%). There was a median of 4 somatic nonsense and missense mutations per exome in the 5 exomes, with a strong tendency for more mutations being identified in the exomes with higher coverage and higher blast percentages. Even though only a small number of leukemias was analyzed by WES, two leukemia exomes had recurring mutations in the same gene (4930595M18Rik), and two other leukemias had mutations in known leukemia drivers involved in cellular proliferation pathways. One leukemia carried an activating mutation in the tyrosine kinase domain of Flt3, and in another exome a mutation in the catalytic domain of the intracellular protein tyrosine phosphatase Ptpn11 was found. PTPN11 is a downstream effector of the Ras pathway and mutations in PTPN11 is repoted in juvenile myelomonocytic leukemia (JMML) and Noonan sydnrome. There was no obvious correlation between the mutations and the type of leukemia (myeloid or myeloid with B220 expression) observed in the mice.
Our results strongly suggest that leukemia only develops if CALM/AF10 is expressed in hemaptoietic stem cells. Expression of CALM/AF10 in B cells is not sufficient for transformation. Presumably, the expression of CALM/AF10 in long-lived hematopoietic stem cells allows for the acquisition of additional, cooperating mutations, which are required for full leukemic transformation.The reproducibility and relatively long latency of leukemia development in the Vav-Cre/R26LSLCA mice should make them a good model for the study of clonal evolution and collaborating events in leukemogenesis.

Disclosures
No relevant conflicts of interest to declare.