Leukemic Stem Cells and Progenitors Demonstrate Impaired Interaction with the Hematopoietic Microenvironment in Vivo in An Inducible Murine Model of Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a stem cell malignancy induced by p210 BCR-ABL and characterized by myeloproliferation in BM and egression of leukemic stem cells and progenitors (LSC/P) to extramedullary sites. Persistence of BCR-ABL+ HSC in patients under Imatinib suggests inhibition of ABL-kinase alone is not sufficient to eliminate the LSC/P. One of the major hallmarks of CML induced by signaling downstream BCR-ABL is the loss of control of the hematopoietic microenvironment on LSC/P. Expression of p210 BCR-ABL has been associated with loss of adhesion to the bone marrow, impaired migration in response to CXCL12 and decreased retention in the BM. In order to study the putative LSC/P niches in steady-state chronic-phase leukemia, we have analyzed the ability of LSC/P to proliferate and get retained in the bone marrow (BM) in an inducible model of CML. Binary transgenic SCL-tTA/TRE-BCR-ABL mice (Koschmieder S et al ., Blood 2005) express p210 BCR-ABL in LSC/P upon doxycycline withdrawal (CML mice). Induced myeloproliferation was associated with activation of the downstream signaling effectors CrkL and p38-MAPK and expansion of circulating (Table 1) and splenic LSC/P but not in BM, suggesting massive LSC/P egression from the marrow (Table 2). Proliferation analysis showed that myeloid expansion in the spleen was secondary to increased cycling of Lin−Sca1+c-Kit+ (LSK) cells (3.1-fold increase in S-phase cells, P <0.05), but not in Lin−/c-Kit+ (LK) population, compared with the control spleens. In agreement with the LSC/P BM content data, the frequency of BM-derived LSK and LK cells incorporating BrdU in CML and in control mice remained similar, suggesting a specific egression of LSC/P from the BM to extramedullary sites. To test whether this model truly represented a model of BM LSC/P egression, we compared the splenic and BM LSC/P compared with their controls regarding their adhesion molecule expression, interaction with the hematopoietic microenvironment (HM) and homing to the overall marrow cavity and endosteal space. Splenic, but not BM-derived, LSK and LSK CD34+ ST-HSCs had increased cell surface expression of CD44 compared to controls (1.35 fold, P =0.006 and 1.23 fold, P <0.05 respectively) and decreased expression of L-selectin (8.7 fold, P <0.05) while expression of CXCR4, α 4 β 1 and α 5 β 1 integrins remain similar in bone marrow and splenocytes from CML and control mice. CML BM progenitors also showed 18-fold reduced adhesion to fibronectin and 1.4-fold increased migration towards CXCL12 compared to control BM progenitors. Myeloproliferative disease was transplantable into non-transgenic littermates and homing of CML BM progenitors was increased (4.3 fold, P <0.005) in myeloablated littermate recipient BM. However, lineage-negative leukemic BM-derived cells which had increased homing in BM of recipient mice had an impaired ability to migrate to the BM endosteal space compared with their littermate controls (control: 31 ± 18% vs CML mice: 17.6 ± 17%), suggesting an specific impairment to lodge in specialized anatomically-defined hematopoietic “niches”. Altogether, this murine model may represent an adequate in vivo system to analyze the ability of p210 BCR-ABL-expressing LSC/P to interact with BM niches and study the control of the hematopoietic microenvironment on LSC/P survival, proliferation and retention. Table 1 Increase in circulating LSC/P in the CML mice after withdrawal of doxycyclin | Peripheral Blood | LSK (×103)Cells/mL Blood P <0.05 | LT-HSC(×103)Cells/mL Blood P <0.05 | CFU-GM+BFU-E/mL Blood P <0.05 | |:----------------:| -------------------------------- | ---------------------------------- | ----------------------------- | | Control | 1.56 ± 0.25 | 0.459 ± 0.29 | 60.86 ± 51.09 | | CML mice | 3.56 ± 1.52 | 2.159 ± 2.03 | 869.6 ± 628.4 | Table 2. Immunophenotypic analysis of BM and splenocytes in control and CML mice | Population | BM (Cells ×104) ( Control ) | BM (Cells x104) ( CML ) | SP (Cells ×104) ( Control ) | SP (Cells x104) ( CML ) | |:------------------:| --------------------------- | ----------------------- | --------------------------- | ----------------------- | | C-Kit + Sca1 + | 24.3 ± 9.9 | 21.3 ± 11 | 6.8 ± 4.5 | 30.1 ± 12.3 ( P <0.05) | | Mac1 + Gr1 + | 1779 ± 307 | 1583 ± 265 | 78.4 ± 32 | 608 ± 377 ( P <0.05) | | CFU-C/10 5 Cells | 342 ± 66 | 334 ± 99 | 63.3 ± 7.09 | 79 ± 6.54 ( P <0.05) |