Abstract
Abstract 1420
Poster Board I-443
As one of the most common genetic alterations found in acute myeloid leukemia (AML), constitutive activation of the FMS-like tyrosine kinase (FLT3) has provided a promising candidate for small molecule targeted therapy. However, the results of FLT3 inhibitor monotherapy trials indicate FLT3 inhibition alone is insufficient to induce consistent and durable responses. Moreover, after an initial response, many patients relapse, suggesting that leukemia-initiating stem cells may be escaping inhibitor-induced cytotoxicity. Currently, the exact stage at which activating mutations in FLT3 occur during transformation is unknown. While FLT3 knockout mice have minor defects in hematopoiesis, very little is known about either the effect of FLT3 activating mutations on normal hematopoietic stem cells or the contribution of FLT3 activation to leukemogenesis. Thus, in order to better understand the underlying molecular mechanisms of transformation and to identify novel targets for treatment of AML, the role of FLT3 activating mutations in hematopoietic stem cells (HSCs) is of great interest.
To study the natural stem cell reservoir and other populations that may escape inhibition, our laboratory has developed a knock-in mouse model in which the FLT3/ITD mutation (an internal tandem duplication correlated with poor prognosis in patients) has been introduced under the endogenous promoter, resulting in myeloproliferative disease (MPD). Conventional transplantation using unfractionated or lineage-depleted marrow from FLT3/ITD mice failed to fully engraft or recapitulate disease, suggesting a HSC defect. Thus, in order to identify a compartment enriched for MPD-initiating cells, several cell surface marker-defined hematopoietic populations were transplanted and compared for engraftment and disease recapitulation. Lineage negative (LIN-), KSL (KIT+SCA+LIN-), MPP (KSL CD34+FLT3+), and ST-HSC (KSL CD34+FLT3-) cells sorted from FLT3/ITD bone marrow all had significantly reduced reconstitution capacity compared to the same compartment from WT littermates. In contrast, highly purified LT-HSCs (KSL CD34-FLT3-) generated equivalent engraftment whether from WT or ITD bone marrow. Furthermore, we measured Hoechst dye efflux in WT and ITD bone marrow to examine side population (SP) cells, known to be enriched in HSC activity, and found that FLT3/ITD mice displayed five-fold fewer SP cells. In addition, bone marrow from FLT3/ITD mice showed a ten-fold decrease in SLAM-defined stem cell frequency (LIN-CD48-CD41-CD150+). 500 sorted SLAM cells from either WT or FLT3/ITD mice were sufficient to fully reconstitute a transplant recipient, demonstrating an equivalent engraftment capacity within this HSC-enriched compartment. Moreover, the MPD phenotype was successfully recapitulated in primary transplants of FLT3/ITD SLAM cells as characterized by an increase in myeloid progenitors, expansion of the LIN- fraction, enlarged spleens and depletion of the SLAM compartment compared to WT SLAM transplant recipients.
Classically defined as a class II oncogene, FLT3 activating mutations have been shown to drive proliferation in cells harboring the mutation. To investigate whether FLT3/ITD drives proliferation in the most primitive hematopoietic compartments, BrdU incorporation was examined in FLT3/ITD hematopoietic stem and progenitor-enriched subsets. While myeloid progenitor compartments showed a decrease in proliferation as compared to WT littermates, the FLT3/ITD SLAM and KSL compartments had an increased percentage of BrdU-incorporating cells. Altogether, our data suggests a role for FLT3/ITD in driving normally quiescent HSCs to proliferate, thereby depleting the pool of primitive HSCs. In this model, HSC depletion coupled to rapid expansion in progenitor cell numbers leads to perturbation of normal hematopoiesis giving rise to a myeloproliferative disease.
Disclosures:
No relevant conflicts of interest to declare.
Maternal exposure to fine particulate matter during pregnancy induces progressive senescence of hematopoietic stem cells under preferential impairment of the bone marrow microenvironment and aids development of myeloproliferative disease
