Inactivation of the Cell Fate Determinate Llgl1 (Lethal Giant Larvae Homolog 1) Leads to Enhanced HSC Fitness and Predicts a Poor Prognosis in AML.

Abstract 2293

Regulation of the balance between symmetric and asymmetric cell division is considered to be important for hematopoietic stem cell (HSC) division and homeostasis. Recently, several polarity regulators have been shown to influence HSC function using RNAi-screening approaches. Llgl1 is a known regulator of cell polarity in drosophila and epithelial cells, however, its role in HSC remains elusive. Using a previously published conditional knockout mouse model with exon 2 of Llgl1 being flanked by loxP-sites we generated Llgl1^loxP/loxP Mx-Cre⁺ animals and induced deletion of Llgl1 (Llgl1^−/−) by administration of pIpC as determined by PCR.

After deletion of Llgl1 no significant abnormalities in hematopoiesis were evident for 16 weeks in the peripheral blood. However, bone marrow of Llgl1^−/− animals revealed up to a 2-fold increase in HSC (CD34^lo Flk2⁻ LSK). When transplanted competitively into recipient animals Llgl1^−/− cells showed a significant competitive advantage compared to Llgl1^+/+ controls, and that competitive advantage increased over time in serial transplants. In tertiary recipient mice the bone marrow was dominated by Llgl1^−/− cells with a chimerism of above 90%. Llgl1 deleted animals show increased numbers of cycling HSC (CD34^lo LSK) when compared to their wildtype counterparts as detected by Ki-67 staining (64.94% ± 2.537 Llgl1^+/+ HSC vs 50.81% ± 4.213 Llgl1^−/− HSC in G0; p=0.0184*) and BrdU incorporation and this effect was restricted to the HSC compartment. The Llgl1 deleted cells proved to be better able to reconstitute hematopoiesis after being stressed in ex vivo culture, and protected the survival of recipient mice to a higher extent than wildtype cells. Gene-expression analysis on sorted Llgl1^−/− versus Llgl1^+/+ steady state HSC revealed known regulators of HSC self-renewal capacity (such as Hox-genes and self-renewal associated transcription factors) to be among the top regulated downstream targets.

The observed phenotype suggests that Llgl1 expression might influence leukemogenesis and its close homologue Llgl2 has been described recently as an early hit in malignant transformation of chronic neutropenia. Thus, we analyzed Llgl1 gene-expression in human acute myeloid leukemia (AML). Llgl1 was the top polarity regulator to be down-regulated in leukemia initiating cells of different phenotype (L-MPP or L-GMP), when compared to their normal counterparts (MPP or GMP). Moreover, reduced Llgl1 expression was associated with inferior overall survival in two independent patient cohorts treated for AML: Low expression of Llgl1 as measured by Affymetrix gene arrays was associated with decreased survival in 83 karyotypic normal (CN-) AML patients below the age of 60 (p=0.0092**). This finding could be confirmed in an independent CN-AML cohort (n=80, p=0.0056**) by qRT-PCR. Moreover, the Llgl1^−/− GEP signature obtained from murine HSC was not associated with a specific genetic subgroups of a human AML dataset, but AML cases matching this signature showed again decreased overall survival (p=0.0089**).

Genetic inactivation of Llgl1 contributes to fitness and viability of hematopoietic stem cells in vivo. Given its impact on survival in patients treated for AML it is tempting to speculate on its role in development and maintenance of leukemia stem cells (LSC).