Group members: Ronan Quéré, Marianne Bussière, Göran Karlsson, Silja Andradottir and Stefan Karlsson
Identify the molecular pathogenesis of leukemias induced by HOXA10, HOXA9 and NUP98-HOXA9.
Homeobox (HOX) genes are important for the regulation of hematopoiesis controlling proliferation, differentiation and stem cell self-renewal (1-3). Overexpression studies with HOXB4 show that hematopoietic stem cells can be greatly expanded in vitro (3), while HOXA9 and HOXA10 are both associated with acute myeloid leukemia (AML) (3). Interestingly, the latency period for malignant transformation is several months, indicating that additional genetic changes are required to generate leukemia. Notably, the latency period can be dramatically reduced from 20-50 weeks to 8-10 weeks if Meis1, a known HOX protein co-factor, is overexpressed together with HOXA9 or HOXA10 (4, 5).
We have investigated how HOXA10 governs normal and malignant hematopoiesis using inducible HOXA10 mice. HOXA10 transgenic mice (6) were crossed with Rosa26-rtTA-nls mice to generate a mouse model where the expression level of HOXA10 can be tightly regulated using doxycycline. The expression of HOXA10 induced an increase in HSCs repopulating capacity in vitro and identified the stem cell regulators Hepatocyte Leukemia Factor (HLF) and Dickkopf 1 (Dkk1) as downstream targets of HOXA10. Notably, the proliferation induction of HSC by HOXA10 was dependent on the HOXA10 concentration, since high levels of HOXA10 had no effect on HSC proliferation. Furthermore, high levels of HOXA10 lead to an accumulation of Megakaryocyte-Erythroid Progenitors (MEP) and blocked erythroid and megakaryocyte development. The block in erythropoiesis and megakaryopoieis is accompanied by downregulation of Gata-1, suggested here as a direct downstream target of HOXA10. Our findings demonstrate that HOXA10 acts as an important regulator of hematopoiesis governing both proliferation and differentiation of hematopoietic progenitor and stem cells, where distinct fate outcomes depend on the HOXA10 concentration.
Engineering of mice with regulated expression of HOXA10. To generate inducible leukemia bone-marrow cells are transduced with a meis1 retroviral vector and transplanted to irradiated recipients. To induce leukemia the recipients are given doxycycline.
To study the role of HOXA10 in malignant transformation overexpression of HOXA10 together with Meis1 will cause malignant transformation to generate AML with a relatively short latency period. By inducing expression of the HOXA10 transgene, we can ask whether malignant transformation is reversible upon withdrawal of HOXA10 overexpression. This in turn, will allow us to define the molecular mechanisms that cause HOXA10 induced leukemia to identify new possible targets for therapy. Our preliminary findings demonstrate that continuous overexpression of HOXA10 is required to generate AML in primary recipient mice, but is not essential for maintenance of the leukemia. Transplantation of AML to secondary recipients shows that in established leukemias, ~80% of the leukemia-initiating cells (LICs) in bone marrow stopped proliferating upon withdrawal of HOXA10 overexpression. However, the population of LICs in primary recipients is heterogeneous since ~20% of the LICs induce leukemia in secondary recipients despite elimination of HOXA10 induced overexpression. Intrinsic genetic activation of several proto-oncogenes was observed in relapsed leukemias. Interestingly, high levels of the adhesion molecule CD44 on LICs are essential to generate leukemia after removal of the primary event. This suggests that extrinsic niche-dependent factors are also involved in the host-dependent outgrowth of leukemias after withdrawal of HOXA10 overexpression event that initiates the leukemia.
Leukemia stem cells (LSCs) are capable of limitless self-renewal and are responsible for the maintenance of leukemia. Since elimination of LSCs will be of therapeutic benefit, it is important to identify regulatory pathways that control their development. We are studying LSCs in a Smad4-/- mouse model of acute myelogenous leukemia (AML) induced either by the HOXA9 gene or by the fusion oncogene NUP98-HOXA9 and have discovered that loss of SMAD4 accelerates the development of leukemia due to an increase in transformation of hematopoietic stem- and progenitor cells (HSPCs). The findings show that HOXA9/SMAD4 complexes accumulate in the cytoplasm of normal HSPCs transduced with HOXA9 or NUP98- HOXA9. In contrast there is no cytoplasmic accumulation of HOXA9 in SMAD4-/- HSPCs and as a consequence increased levels of HOXA9 accumulate in the nucleus leading to increased bone marrow transformation to leukemia. Therefore, the cytoplasmic sequestration of SMAD4 by high levels of HOXA9 is a mechanism that facilitates HOXA9-induced transformation of normal HSPCs. Since SMAD4 is a potent tumor suppressor involved in growth control, we developed a strategy to restore the subcellular distribution of SMAD4 through counteracting the sequestration introduced by high levels of HOXA9 in HSPCs. We successfully disrupted the interaction between HOXA9 and SMAD4 to reactivate the TGF-? pathway in HSPC, leading to a loss of LSCs with subsequent prevention/treatment of leukemia. Together, these findings reveal a major role for SMAD4 in the negative regulation of leukemia initiation and maintenance induced by HOXA9/NUP98- HOXA9 and provide strong evidence that antagonizing Smad4 sequestration by these oncoproteins might be a promising novel therapeutic approach in leukemia.
The figure above shows interactions between fusion oncoproteins TEL-AML, AML-EVII and Nup98-HoxA9 and Smads (TEL-AML1 [Ford et al., 2009], AML-EVI1 [Kurokawa et al., 1998], Nup98-HoxA9 [our own studies]).
The figure above shows a confocal microscopic analysis that demonstrates association between Smad4 and HoxA9. These and other data show that Smad4 is sequestrated to the cytoplasm by HoxA9 and Nup98-HoxA9 (Quéré et al, submitted for publication).
• Prof. R Keith Humphries, Terry Fox Laboratory, BC Cancer Research Center, Vancouver
• Prof. Jeffrey H Lawrence, Hematology Research, VAMC, San Francisco, CA
• Dr. Jörg Cammenga, Lund University.
• Prof. Roman Zubarev, Karolinska Institute, Stockholm
• Prof. Thoas Fioretos, Lund University and International Clinical colleagues.
Page Manager: Jens Forsberg
Last modified: 2010-07-30
