HoxA9 Knockdown Inhibits Proliferation and Induces Cell Death in Human MLL-Rearranged Leukemias.
Authors: Faber Jorg , Krivtsov Andrei V. , Stubbs Matthew C. , Wright Renee , van den Heuvel-Eibrink Marry , Kung Andrew L. , Zwaan Christian M. , Armstrong Scott A. 
Published: April 2020

Abstract
Homeobox containing (Hox) genes are implicated in the regulation of normal and leukemic hematopoesis. Using gene expression profiling we and others have previously shown that HoxA9 is highly expressed in lymphoid and myeloid leukemias harboring MLL translocations and that high level HoxA9 expression is associated with poor clinical prognosis. Furthermore, HoxA9 plays variable roles in MLL-fusion induced murine leukemias. In this study we aimed to elucidate the role of aberrant HoxA9 expression in human MLL-rearranged and non-rearranged leukemia’s utilizing an shRNA mediated knockdown approach. To establish an efficient knockdown assay three different shRNA constructs targeting human HoxA9 were synthesized and stably introduced into t(9;11) MOLM14 cells utilizing a lentiviral vector system. The shRNA construct which showed highest efficiency as measured by Taqman quantitative PCR (75–80% knockdown MLL-AF9 RNA) and Western Blot analysis was used for further experiments. In MOLM-14 cells, HoxA9 directed shRNA inhibited cell proliferation starting as early as 48h after transduction as determined by MTT assay, and at 72h demonstrated a markedly increased number of apoptotic cells as measured by Annexin V staining. This effect was rescued by introducing a non-targetable exogenous HoxA9 in MOLM-14 cells. To investigate if the HoxA9 knockdown related effects are specific for MLL rearranged cells we next analyzed cell growth and viability in 17 AML/ALL cell lines (7 MLL-rearranged, 10 non rearranged) after shRNA mediated HoxA9 knockdown. Interestingly, impaired cell proliferation and induction of apoptosis was significantly higher in the MLL rearranged cell lines (mean viability: 51.88%) than in the non-rearranged cells (mean viability: 90.98%; p=0.007). Moreover, the effect was also significantly correlated with the baseline HoxA9 mRNA expression before knockdown, with the greatest effect in cell lines expressing the highest HoxA9 levels (R= 0.8, p=0.00017). These findings prompted us to further analyze the effect of HoxA9 knockdown in MLL rearranged and non-rearranged primary human AML cells (6 MLL rearranged, 6 MLL germline). Similar to our findings in cell lines, we found a significantly higher effect on cell proliferation/viability in association with the presence of an MLL translocation (p=0.005) and a significant correlation with the baseline HoxA9 mRNA expression (R= 0.8, p=0.001). Next, we assessed the in vivo effect of HoxA9 knock down by transplanting luciferase-expressing SEMK2 (t4;11) cells and subsequent bioluminescent imaging. SEMK2 cells were transduced with either HoxA9 directed or control shRNA and intravenously injected into SCID-beige mice.
Reconstitution was confirmed by in vivo bioluminescent imaging. 34 days after transplantation all mice in the HoxA9 shRNA group (n=4) are still alive with no signs of leukemia whereas all mice in the control group (n=3) have succumbed. Taken together our data implicates an important role for aberrant HoxA9 expression in human MLL rearranged leukemia cells.