HOX A9: Connections to Acute Myeloid Leukemia Doug Ornoff BIOL

Summary  Acute myeloid leukemia (AML) is a cancer of the bone marrow that produces WBCs, and results in a proliferation of nonfunctional leukocytes that interfere with normal blood cell function.  HOXA9 normally functions in skeletal patterning along the anteroposterior (A-P) axis, and in proper natural hematopoiesis.  Various chromosomal translocations result in either upregulation of the HOXA9 gene or creation of a fusion protein.  Result is a transcriptional increase in hematopoietic proliferative proteins and a decrease in proteins promoting leukocyte differentiation  Future treatment options lie in regulating cofactors necessary for HOXA9 function.

Acute Myeloid Leukemia (AML)  A leukemia, thus affecting the blood and blood- forming organs  Leukemias cause an abnormal increase in the number of leukocytes, specifically the neutrophils, eosinophils, basophils, and monocytes  Results in a population of immature white blood cells that fail to respond to death signals, accumulate, spill out into the blood, travel to other areas, and interfere with normal cell function. Above: Normal human leukocytes. Univ. of Western Australia Below: Leukocytes from bone marrow of AML patient. UVA Medical.

AML Symptoms  Pallid complexion  Anemia, due to disrupted erythrocyte function  Mild fever, frequent mild infections  Prolonged bleeding from gums and from minor cuts due to platelet disruption  Fatigue and shortness of breath  Excessive bruising and spotting of the skin

Hematological Signs of AML  Crowding of normal bone marrow by proliferating nonfunctional WBCs  Anemia, as evid. by low hemoglobin & hematocrit  Neutropenia / granulocytopenia (not enough granulocytes), as evid. by increased susceptibility to normal infections  Thrombocytopenia (too few platelets)  Shortage of normal WBC (leukopenia), but high numbers of nonfunctional undifferentiated WBCs (leukocytosis)

Current Treatment Options  Surgery not an option, since leukemia cells not confined to any one body region  Chemotherapy utilizes drugs aimed at disrupting cell division; affects diseased bone marrow and mutated hematopoietic cells especially  Common chemo drugs are cytarabine (ara-C) and an anthracycline, such as daunorubicin or idarubicin.  Bone marrow or peripheral blood stem cell transplantation used to recover hematopoiesis  Immunotherapy using monoclonal antibodies targeted at AML cells becoming useful, and new methods use Abs with radioactive or toxic groups attached.

Normal HOXA9 Function – Skeletal Patterning Fromental-Ramain et al.  HOXA9 is involved in mice in skeletal patterning and development along the anteroposterior (A-P) axis.  Double mutant phenotype features supernumerary ribs, as well as anteriorization of the lower thoracic and lumbar and saccral vertebrae.

Normal HOXA9 Function – Skeletal Patterning Fromental-Ramain et al.  Murine vertebrae Left column: HOXA9 -/- Right column: WT

HOXA9 at the subcellular level  First and foremost, it’s a transcription factor!  Interactions with cofactors Meis1 and Pbx1 help recruit other coactivators or corepressors that turn on/off genes  One important role involves the coactivator CBP, which allows CREB to bind and stimulate RNA PolII Above: HoxA9 (H) recruiting coactivator (CoA) along with cofactor (Co). Below: HoxA9 (H) recruiting corepressor (CoR) or inhibiting coactivator (CoA). Owens et al. Stem Cells 2002; 20:

Normal HOXA9 Function –Hematopoiesis  Within the organism, HOXA9 is further involved in hematopoiesis, playing a role in the regulation of both blood stem cell proliferation AND differentiation. Gilliland, Gary. “Molecular Paradigims/Mechanisms in Acute Myeloid Leukemia.” State of the Science Leukemia Conference. National Cancer Institute, 2000

Normal HOXA9 Function –Hematopoiesis Dr. Thomas Graf, Albert Einstein College of Medicine  Proliferation, followed by differentiation, produces mature functional leukocytes.

Normal HOXA9 Function –Hematopoiesis Lawrence et al.  Lawrence et al constructed knockout mice and noted that the mice had disrupted hematopoiesis and lower numbers of WBCs, lymphocytes, and granulocytes

Abnormal HOXA9 Gene Function  Abnormal gene function is almost always related to a chromosomal translocation  The translocation affects HOXA9 gene in one of three ways: upregulation due to MLL overexpression overexpression due to promoter placement overlay resulting in fusion protein  EX. HOXA9-NUP98 fusion

Progression to Cancer – Human/Mouse Homologies in HOXA9 Mus musculus (top) Homo sapiens (bottom) 1 mattgalgny yvdsfllgad aadelgagry apgtlgqppr qaaAlaehpd fspcsfqska 1 mattgalgny yvdsfllgad aadelsvgry apgtlgqppr qaaTlaehpd fspcsfqska 61 avfgaswnpv haaganavpa avyhhhhhpy vhpqapvaaa apdgrymrsw leptpgalsf 61 tvfgaswnpv haaganavpa avyHhhhhhpy vhpqapvaaa apdgrymrsw leptpgalsf 121 aglpssrpyg ikpeplsarr gdcptldtht lsltdyacgs ppvdrekqps egafsennae 181 nesggdkppi dpnnpaanwl harstrkkrc pytkhqtlel ekeflfnmyl trdrryevar 241 llnlterqvk iwfqnrrmkm kkinkdrakd e

Progression to Cancer – HOXA9 and Pbx1

Progression to Cancer – NUP98-HOXA9 fusion  Kroon et al. went on to demonstrate that mice transfected with bone marrow containing a NUP98-HOXA9 fusion were induced into an AML phenotype.  They also found that when fusion transfection was done alongside overexpression of Meis1, AML onset was accelerated.

Progression to Cancer – An Rb Analogy

Structural Insights  Further structural studies on a HOXA9/Pbx1/DNA complex by LaRonde-LeBlanc et al. revealed that the HOXA9/Pbx1 pair binds to DNA with high affinity  Binding is stronger than other HOX/DNA complexes due to major and minor groove interactions.  DNA recognition sequences are also embedded in the HOXA9/Pbx1 structure. HOXA9 and PBX 1 bound to DNA. 1.90A. RCSB Protein Data Bank

Structural Insights  Additionally, Kasper et al found that the 38 conserved FG repeats in a domain of NUP98 were responsible for CBP binding and subsequent recruitment of CREB.  Their studies showed that in fusion with HOXA9, the FG repeats were preserved.  Interestingly, the fusion results in a deletion of a portion of the HOXA9 N-terminus that has transcription repression properties.

Current Research and Future Treatment Options  Further elucidate the interaction between the NUP98-HOXA9 fusion and Meis1  Understand the role between HOXA9 expression and MLL  Continue investigations on the Core Binding Factor subunit (CBFα, aka AML1, and CBFβ) (CBF recruits CRB, which recruits CREB)  The large number of ways that the HOXA9 pathway can become improperly activated also provides a large number of ways that it can be therapeutically targeted.  Work is underway to try to disrupt the co-activator and co- repressor systems.  Perhaps another option is to consider disrupting the HOXA9/DNA binding, or to hyperactivate the differentiation pathway.

Summary  Acute myeloid leukemia (AML) is a cancer of the bone marrow that produces WBCs, and results in a proliferation of nonfunctional leukocytes that interfere with normal blood cell function.  HOXA9 normally functions in skeletal patterning along the anteroposterior (A-P) axis, and in proper natural hematopoiesis.  Various chromosomal translocations result in either upregulation of the HOXA9 gene or creation of a fusion protein.  Result is a transcriptional increase in hematopoietic proliferative proteins and a decrease in proteins promoting leukocyte differentiation  Future treatment options lie in regulating cofactors necessary for HOXA9 function.

References American Cancer Society. “Acute Myeloid Leukemia.” Cancer Reference Information. Leukemia and Lymphoma Society. “Acute Myeloid Leukemia.” Disease Information. Cancer Genetics Web. HoxA9 lookup. Gilliland, Gary. “Molecular Paradigims/Mechanisms in Acute Myeloid Leukemia.” State of the Science Leukemia Conference. National Cancer Institute, Owens, Bronwyn M. and Hawley, Robert H. HOX and Non-HOX Homeobox Genes in Leukemic Hematopoiesis. Stem Cells 20: Fromental-Ramain, Catherine et al. Specific and redundant functions of the paralogous Hoxa-9 and Hoxd-9 genes in forelimb and axial skeleton patterning. Development 122, Kasper, LH. et al. CREB binding protein interacts with nucleoporin…Mol Cell Bio. 1999, 19: LaRonde-LeBlanc et al. Structure of HoxA9 and Pbx1 bound to DNA: Hox hexapeptide and DNA recognition anterior to posterior. Genes and Development 17: , 2003 Lawrence, HJ et al. Mice bearing a targeted disruption… Blood 1997;89: Nakamura et al. Cooperative activation of HOXA and Pbx1-related genes in murine myeloid leukemias. Nat Genetics 1996;12: Kroon E et al. NUP98-HOXA9 expression in hematopoietic stem cells induces chronic and acute myeloid leukemias in mice. EMBO J 2001;20;