H2.0-Like Homeobox (HLX) Causes Pre-Leukemic Myeloid Expansion and Initiates AML In Cooperation With FLT3-ITD

Acute myeloid leukemia (AML) is an aggressive disease associated with poor clinical outcome. Less than one third of patients achieve durable remission with current treatment regimens, and prognostication and risk stratification are challenging. We have recently reported that the non-clustered homeobox gene, H2.0-like homeobox (HLX), is 2 to 16 fold overexpressed in more than 80% of patients with AML, across all major disease subtypes, and higher levels of HLX are associated with poor overall survival in AML. Inhibition of HLX in both murine and human AML cells has a significant anti-leukemic and differentiation-inducing effect suggesting HLX and its downstream targets as novel therapeutic targets in AML.

In order to better understand the role of Hlx at the stem cell level and in myeloid differentiation in vivo, we generated knock-in mice conditionally overexpressing Hlx from the Rosa26 locus and bred them to mice that bear Cre recombinase under the control of the pIpC-inducible, hematopoietic specific promoter, Mx1. Animals overexpressing HLX exhibit elevated WBC counts and abnormal myeloid cells in the peripheral blood. Analysis of the bone marrow reveals expansion of the granulocyte-macrophage progenitor population (lin- ckit+ cd34+ CD16/32^high) and expansion of immature myelocytes (ckit+ cd34+ CD16/32^high Gr1^int). Hlx knock-in bone marrow cells, and specifically immature granulocyte precursors, exhibit enhanced serial clonogenicity in methylcellulose colony assays, and a differentiation block and maintenance of immaturity in response to GM-CSF.

Internal tandem duplications of FLT3 (FLT3-ITD) are seen in approximately 30% of all AML patients, and frequently co-occur with elevated HLX levels. Correlative analyses showed that AML patients with mutant FLT3 and low HLX have overall survival similar to WT FLT3 patients, and survive significantly longer than patients with mutant FLT3 and high HLX (p=0.005), demonstrating that FLT3 mutations confer poor prognosis only if HLX is highly expressed, and suggesting that HLX and mutant FLT3 functionally cooperate. We retrovirally co-expressed HLX and FLT3-ITD, or FLT3-ITD alone (plus an empty control), in primary Lin^-Kit⁺cells and transplanted them into congenic recipient animals. Four weeks after transplantation, donor chimerism was 4-fold increased on average in the peripheral blood (PB) and bone marrow (BM), and by 12 weeks post-transplantation mice expressing FLT3-ITD and HLX developed AML with large numbers of leukemic blasts in the peripheral blood and bone marrow.

We then crossed our new Hlx knock-in mouse model with previously generated FLT3-ITD knock-in mice. Strikingly, heterozygous double-transgenic mice expressing both the knock-in FLT3-ITD mutation and HLX develop acute myeloid leukemia after a latency of 2 months. Morphological and flow cytometric analysis revealed large numbers of blasts circulating in the peripheral blood and replacing the marrow, as well as substantial leukemic infiltrates in the spleen and liver.

Our studies reveal a critical role for HLX in conferring a differentiation block and increased clonogenicity at the pre-leukemic stem and progenitor cell level in a genetic in vivo model. Furthermore, a novel compound knock-in mouse model of Hlx overexpression and FLT3-ITD demonstrates that Hlx can initiate AML in cooperation with FLT3-ITD in vivo.