The role of activating mutations of receptor tyrosine kinases (RTKs) in hematopoietic malignancies is well-established. PDGFβR is one such RTK that is a frequent target in myeloid leukemias. More than fifteen different translocations have been identified in human CMML patients that lead to constitutively active fusion variants of PDGFβR (Curtis et al., 2007
). Accumulating evidence suggests that all PDGFβR-containing translocations share many properties and are functionally interchangeable. For example, one such translocation, t(5;7)(q33;q11.2), fuses the majority of the HIP1
gene to the transmembrane and catalytic domains of the PDGFβR
gene (Ross et al., 1998
). Like all of the other PDGFβR fusions, the H/P fusion protein is a constitutively active tyrosine kinase that is able to transform hematopoietic cells to factor-independent growth in culture (Ross et al., 1998
) and activate the PI3K, MapK, PLC-gamma and STAT5 pathways (Ross and Gilliland, 1999
). The role of these oncogenes in vivo
has only been studied using the retroviral bone marrow transduction and transplantation assays. In the current study, we employed a previously described knockin trategy (Higuchi et al., 2002
) that couples a loxP
bracketed transcriptional stop cassette with an inducible Cre transgene to generate a mouse in which lineage-specific expression of H/P can be temporally and spatially controlled. This strategy allows us to bypass any embryonic lethality that results from the expression of H/P (Oravecz-Wilson et al., 2004
) and to directly determine the development of disease in the adult mouse in the presence or absence of other genetic lesions such as a conditional A/E knockin allele (Higuchi et al., 2002
Following the generation of the Mx1-Cre;Hip1+/LSL-H/P
mice, we found that these mice did not frequently develop hematopoietic malignancies following pIpC induction even after 1.5 years. H/P expression is, therefore, not sufficient to cause cancer, though a minority of mice did exhibit enlarged spleens and liver tumors suggesting that H/P expression increases the risk of carcinogenesis. This finding contrasts starkly with BTT assays where retroviral expression of tyrosine kinase oncogenes such as H/P (Figure S1
), TEL/PDGFβR (Grisolano et al., 2003
), and BCR/ABL (Neering et al., 2007
) induced a MPD similar to CML. Importantly, since healthy, non-neoplastic tissues (kidney, liver, spleen) from pIpC-treated Mx1-Cre;Hip1+/LSL-H/P
mice maintained Hip1H/P
alleles (), the possibility that Hip1H/P
-containing cells were selected against over time is an unlikely explanation for the lack of disease after 1.5 years. Since use of endogenous regulatory elements to express the H/P oncogene did not lead to the MPD observed following retroviral expression of tyrosine kinase oncogenes, the cell type and/or oncogene levels are seemingly key variables to control when developing mouse models of human cancers.
In contrast to the finding that expression of H/P alone did not result in transformation of hematopoietic cells in vivo
, co-induction of H/P with A/E led to a florid MPD within days of induction. Thus, contrary to the BTT models of tyrosine kinase oncogene and A/E oncogene cooperation that yield prolonged disease latency and predominantly acute leukemia (Grisolano et al., 2003
; Schessl et al., 2005
), this murine model provides direct evidence that expression of A/E in adult myeloid progenitors in vivo
does not completely block differentiation but instead cooperates with H/P to induce an explosive myeloproliferative neoplasm. Moreover, these data confirm that A/E induced leukemia is a multi-step process, in which secondary genetic alterations, such as H/P fusion protein generation, cooperate with A/E to induce full transformation. This report demonstrates that a human oncogene can indeed cooperate with the A/E knockin allele to produce transformation (Higuchi et al., 2002
While a clear hematologic response to imatinib was observed in these double knockin mice, transplant and drug withdrawal experiments demonstrated that LICs were not eliminated by this therapy. This H/P;A/E disease model highlights the need for therapies that not only target oncoproteins but also preferentially kill leukemogenic cells. The mechanism(s) of resistance of LICs to imatinib therapy is not yet understood, but possibilities such as limited intracellular drug levels in the LIC, limited expression of the oncogene, relative quiescence, or lack of addiction of the LIC to the oncogene are all currently under investigation. It will be important to determine if the LICs depend upon the initiating H/P oncogene signals for survival. If the LIC is not dependent upon- or cannot be manipulated to be dependent upon the H/P activity, therapeutic variations on the imatinib therapy theme will be ineffective.
These mice model the neoplastic role of the growing family of PDGFβR human translocations in vivo
more accurately than before by using a conditional knockin technology. Furthermore, these experiments provided intriguing insights into how the different ways used to model leukemia in mice influence outcomes and conclusions. We expected that activation of a single copy of H/P cDNA knocked into the endogenous Hip1
locus in the adult mouse would be sufficient to induce a chronic MPD. In addition, based on the report by Grisolano et al. (Grisolano et al., 2003
), we expected that co-induction of A/E with this H/P allele would result in an increased frequency or severity of the H/P-induced disease and would inhibit myeloid differentiation leading to acute leukemia. To our surprise, however, a very low frequency of neoplasia was observed in the single H/P knockin mice, and a shockingly high frequency (100% penetrance) and an essentially absent disease latency of an explosive MPD (rather than acute leukemia) was observed in the double knockin mice. This observation directly counters the prevailing idea that A/E functions via prevention of hematopoietic differentiation (Reilly, 2003
). If this notion were true, the H/P;A/E knockin mice would be immediately blastic and have impaired differentiation. These findings raise the possibility that modeling leukemogenic events under the most genetically realistic conditions may help us identify environmental or genetic triggers that lead to the development of CML accelerated phase or frank blast crisis.
In sum, the conditional activation of H/P in the hematopoietic system of adult mice leads to an explosive, mature, imatinib-sensitive MPD when co-expressed with A/E. Importantly, LIC activity was persistent in induced H/P;A/E mice following imatinib treatment as their bone marrow was able to transplant this imatinib-sensitive MPD more readily than vehicle treated mice. This model will therefore serve as an invaluable tool for in depth studies of the therapeutic effect of PDGFβR (imatinib, MAPK inhibitors, rapamycin) and A/E (trichostatin A, SAHA) inhibitors as well as allow for further rigorous in vivo characterization of the cellular and molecular mechanisms of hematopoietic transformation and drug resistance.