Identifying the Leukemia Stem Cell Niche Mediating Drug Resistance in Flt3-ITD Acute Myeloid Leukemia

The Flt3-ITD is one of the most common mutations in AML, and defines a distinct subtype of disease with unique features and biology, and with poor prognosis related to high rates of recurrence. Although several FLT3 TKIs have been developed for clinical use, responses are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC drug resistance to Flt3-targeted TKI in Flt3-ITD AML.

We utilized a newly generated Flt3-ITD TET2^flox/flox Mx1-cre mouse model of AML to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. Administration of pIpC leads to deletion of TET2 and development of AML-like disease characterized by leukocytosis, accumulation of blasts, anemia and thrombocytopenia. Transplantation of selected STHSC, MPP and GMP populations revealed that LIC were almost exclusively found within the phenotypic ST-HSC population (calculated stem cell frequencies: <1:180,000, 1:63,635, and 1:2,730 for GMP, MPP, and ST-HSC, respectively). We similarly found that in samples from human Flt3-ITD AML patients LIC capacity was similarly restricted to more primitive HSPC populations (Lin-CD34+CD38-) and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+).

We performed flow cytometry on collagenase-digested bone fragments from AML mice to characterize bone marrow stromal cells in Flt3-ITD AML mice. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found that several stromal populations are expanded in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+), a 3.9-fold increase in endothelial cells (CD45-Ter119-CD31+), and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). The expression of CXCL12, a key factor that mediates niche localization of HSC and LSC, was greater than 2-fold higher in osteoprogenitors, but not significantly different in endothelial cells, and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC.

We transplanted murine AML cells into CXCL12^flox/flox UBC-cre or CXCL12^flox/flox Osx-cre mice to assess the effect of global or osteoprogenitor-specific CXCL12-KO, respectively, on AML progression and TKI response. We found that this model of AML was resistant to single agent Flt3 TKI (AC220) treatment. Global CXCL12-KO using CXCL12^flox/flox UBC-cre mice modestly improved response to TKI. We show that a combination regimen including standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and a Flt3 TKI (AC220) results in more effective and selective targeting of leukemia cells in this model. We are currently treating osteoprogenitor-specific CXCL12-KO AML mice with the combination chemotherapy + TKI regimen to investigate the contribution of osteoprogenitors to disease progression and drug resistance in Flt3-ITD AML LSC.

In conclusion, our results suggest that LSC in Flt3-ITD AML are present within a primitive phenotypic ST-HSC population more so than in MPP and GMP populations as often seen in some other types of AML. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML.