HOXA9 Regulates Enhancers of Genes involved in Hematopoiesis and Leukemia Where does this HOXA9 transcription factor function in the genome? To determine genes controlled by HoxA9 a HoxA9-ER gene was inserted into a retrovirus an then into bone marrow. These cells could only grown in the presence of 4-OHT. RNA was taken from these cells at 72, 96 and 120 hours after 4-OHT was withdrawn and analyzed using Affymetric microarray hybridization. Genes that were upregulated during the expression of HoxA9 and slowly decreased upon its removal are proliferative genes and genes that were down regulated are those specific to myeloid differentiation and inflammatory genes. Using ChIP-Seq anaylsis a genome wide study of HoxA9 binding was performed in this experiment. HoxA9 along with its cofactor was found to bind evolutionarily conserved sites that were very distant for the transcription start site. The binding sites had more enhancer like qualities with high histone H3K4 monomethylation and CBP/P300 binding. The genome wide results suggested that HoxA9 is part of the enhancesome. Binding of HoxA9 is associated with increased acetylation and coactivator recruitment. A variety of oncogenic alterations, including MLL fusion proteins, CDX2 or CDX4 overexpression, NPMc or NUP98 fusion proteins enforce high level Hoxa9 expression, making enhanceosome coactivator activity refractory to physiologic differentiation signals. The resulting persistent expression of proliferative target genes such as Flt3, Sox4, Lmo2 and Myb leads to leukemic transformation.

Knockout HOXA9 Mice have Defects in Hematopoietic Stem Cells To determine the function of HOXA9 mice were made to express nonfunctional HOXA9. Mice were sublethally irradiated and then injected with marrow cells from their siblings. The marrow cells were either Hoxa9 knockout or wild-type. The knockout mice developed pancytopenia due to the inability of their stem cells to repopulate the various blood cells. Hoxa-9 deficient cells have a decreased ability to proliferate. The left graphs confirm that there is difference in proliferative ability between wild-type and mutant Hoxa-9 cell lines. Cells in this experiment were placed in bulk liquid cultures and a mix of the same cytokines as in the left most figure. There was both a decrease in the number of cells in the mutant line culture and number of colonies. In figure B we see how that decrease in proliferation results in decreased numbers of mature blood cells in wild-type versus knockout Hoxa-9 mice. Stem cell function was measured in mutant and wild-type mice at various weeks post x-ray irradiation. The mutant mice developed significant lymphopenia, neutropenia, and anemia. The defect seen in multiple blood cell lineages implies that there is a problem arising in the pluripotent stem cell that is inhibiting the ability to produce lymphocytes, granulocytes and erythrocytes following sublethal irraditation.

A Chromosomal Translocation creates a NUP98-HOXA9 Oncogene NUP98 is protein component of nuclear pore complex and allows for transport between cytoplasm and nucleus The chromosomal translocation, t(7;11)(p15;p15) is found in acute myeloid leukemia patients cancer cells. A piece of chromosome 11 breaks off and fuses to chromosome 7. The resulting gene fusion connects the 5’ part of the NUP98 protein to the 3’ end of the HOXA9.

Overexpressed HOXA9 Increases Concentration of Blast Cells How does the fusion protein behave in cells? Retroviral expression of NUP98-HOXA9 in human cord blood cells lead to decreases in blood cell differentiation, inhibition of erythropoiesis (red blood cell formation), slower neutrophil maturation, and a decrease in myeloid colony formation in response to colony stimulating factors G-CSF and GM-CSF. The transduced human stem cells were examined after 3 weeks and there was a significant increase in immature blood cells and reduction in mature cells in the NUP98-HOXA9 cells in comparison to the control cells. After five weeks of being transduced by the retroviral vectors the NUP98-HOXA9 or NHA9 had much higher Colony Forming Cells (CFC). CFC are representative of progenitor blood cells. CFU-GEMM is colony forming unit granulocyte, erythroid, monocyte, megakaryocyte, CFU-GM is for granulocyte and macrophages and BFU-E is burst forming unit erythroid which is the earliest progenitor erythroid cell. There is an increase in primitive stem/progenitor cells associated with the expression of the NUP98-HOXA9 gene.

Long-term Proliferation and Lack of Differentiation The NUP98-HOXA9 fusion protein results in a NH2-terminal NUP98 attached to COOH-terminal HOXA9. The NUP98-HOXA9 gene was retrovirally transduced to be expressed in CD34+ hematopoietic cells from human peripheral blood in order to study the biological effects of the translocation. In figure A there is an increase in colony number in the NUP98-HOXA9 cells than in the control cells. The control cells contained normal looking red erythroid cells while the mutant cells were large and contained irregular shapes. The Giemsa staining results showed that there more immature blast cells in the experimental cells than in the control which contained mostly mature erythroid precursors. Figure B displays the similar results. Mutant cells proliferated for a greater period of time in this study, around 54.3 days in comparison to the controls cells which stopped growing at around 27.3 days. In figure C flow cytometry analysis was used to support that there was a lack of erythroid differentiation. There is an increase in CD235a+ and a decrease in CD45+ which would be a mark of immature erythroid cells.

Acute Myeloid Leukemia Patients with NUP98-HOXA9 oncogene and decreased remission rates Various genetic mutations in patients with AML can determine whether the prognosis is good or bad. Reverse transcriptase-polymerase chain reaction was used to measure the amount of HOXA9 expression in 54 AML patients and 20 healthy individuals using bone marrow mononuclear cells. HOXA9 expression was found in none of the healthy individuals and in 40.74% of AML patients. AML patients who expressed the gene had a lower remission rate of 45.45% in comparison to AML patients who did not express HOXA9 with 69.70%.

Acute Myeloid Leukemia Presents as Bone Marrow Failure and Hyperleukocytosis Anemia, infection due to neutropenia, bruising and hemorrhage due to thrombocytopenia can all occur due to AML. It is the second most common leukemia diagnosed in adults with a 25.9% survival rate. There is an inability of the committed myeloid progenitor cell to differentiate. Blast cells can be distinguished based on the high nucleus to cytoplasm ratio.

Chemotherapy used to treat Acute Myeloid Leukemia by interference in DNA replication The two most common drugs used to treat AML are Cytarabine and anthracycline drugs which include daunomycin, idarubicin and mitoxantrone. Cytarabine functions as an antimetabolite which inhibits certain phases of the cell cycle. It is similar to cytosine deoxyribose so it is incorporated during DNA replication but quickly converts to cytosine arabinoside triphosphate. This damages the DNA and holds it in S phase. Anthracycline drugs are intercalating agents that inhibit transcription and DNA replication.

Work Cited Li, De-Peng, Zhen-Yu Li, Wei Sang, Hai Cheng, Xiu-Ying Pan, and Kai-Lin Xu. "HOXA9 Gene Expression in Acute Myeloid Leukemia." SpringerLink. Springer US, 12 Apr. 2013. Web. 22 Mar. 2017. Chung, K. Y., G. Morrone, J. J. Schuringa, M. Plasilova, J. H. Shieh, Y. Zhang, P. Zhou, and M. A. Moore. "Enforced Expression of NUP98-HOXA9 in Human CD34(+) Cells Enhances Stem Cell Proliferation." Cancer Research. U.S. National Library of Medicine, 15 Dec. 2006. Web. 22 Mar. 2017. Huret, Jean. "T(7;11)(p15;p15) NUP98/HOXA9." T(7;11)(p15;p15) NUP98/HOXA9. N.p., n.d. Web. 22 Mar. 2017. Lawrence, H. Jeffrey, Julie Christensen, Stephen Fong, Yu-Long Hu, Irving Weissman, Guy Sauvageau, R. Keith Humphries, and Corey Largman. "Loss of Expression of the Hoxa-9Homeobox Gene Impairs the Proliferation and Repopulating Ability of Hematopoietic Stem Cells." Blood. © 2005 by The American Society of Hematology, 01 Dec. 2005. Web. 22 Mar. 2017. Huang, Yongsheng, Kajal Sitwala, Joel Bronstein, Daniel Sanders, Monisha Dandekar, Cailin Collins, Gordon Robertson, James MacDonald, Timothee Cezard, Misha Bilenky, Nina Thiessen, Yongjun Zhao, Thomas Zeng, Martin Hirst, Alfred Hero, Steven Jones, and Jay L. Hess. "Identification and Characterization of Hoxa9 Binding Sites in Hematopoietic Cells." Blood. American Society of Hematology, 12 Jan. 2012. Web. 22 Mar. 2017. Takeda, A., C. Goolsby, and N. R. Yaseen. "NUP98-HOXA9 Induces Long-term Proliferation and Blocks Differentiation of Primary Human CD34+ Hematopoietic Cells." Cancer Research. U.S. National Library of Medicine, 01 July 2006. Web. 22 Mar. 2017.

Works Cited https://link.springer.com/article/10.1007%2Fs12013-013-9586-8 https://www.ncbi.nlm.nih.gov/pubmed/17178874 http://atlasgeneticsoncology.org/Anomalies/t0711p15ID1016.html https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1895111/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257007/ https://www.ncbi.nlm.nih.gov/pubmed/16818636