The NUP98-NSD1 fusion protein, product of the t(5;11)(q35;p15.5) chromosomal translocation, is an AML-associated cytogenetically silent genetic aberration, recently identified as the most frequent fusion in pediatric AML, generally associated with aggressive disease and poor prognosis. Interestingly, the vast majority (>70%) of the reported NUP98-NSD1-positive cases also carried an activating FLT3-ITD mutation suggesting functional cooperation. The purpose of this study was to search for experimental evidence of a functional cooperation between NUP98-NSD1 and FLT3-ITD in the transformation of murine hematopoietic cells in vitro and in vivo.
Lineage surface marker-depleted murine bone marrow cells were transduced with either pMSCV-NUP98-NSD1-neo or pMSCV-FLT3-ITD-GFP or both expression constructs on fibronectin-coated plates. Serial colony formation assays in myeloid favoring medium and immunophenotypic analysis by flow cytometry indicated that retroviral expression of NUP98-NSD1 provided increased self-renewal capacity and impaired differentiation of murine bone marrow stem and progenitor cells.
NUP98–NSD1 expressing cells displayed a typical myeloblastic morphology and co-expressed myeloid and early stem cell surface markers (CD34low/c-kit+/FcgR+/Gr-1+/ Mac-I+/B220-). Co-expression of FLT3-ITD resulted in high rates of cell proliferation, showed a more differentiated phenotype and concomitantly impaired the in vitro clonogenic capacity in methylcellulose cultures.
Bone marrow cells expressing NUP98-NSD1 with or without FLT3-ITD were harvested from methylcellulose cultures and transplanted into sub-lethally irradiated syngeneic mice. All mice receiving cells co-expressing NUP98-NSD1 and FLT3-ITD developed AML that was transplantable into all secondary recipients. Myeloid leukemic blasts that co-expressed NUP98-NSD1 and FLT3-ITD were present in abundance both in BM preparations and in blood smears, and histopathological analysis showed widespread infiltration into solid organs. By contrast, no AML ever developed in mice receiving cells expressing only NUP98-NSD1. These mice, similar to mice receiving cells expressing FLT3-ITD only, developed signs of a chronic myeloproliferative disorder, characterized by expansion of Mac-1+/Gr-1+ BM cells with granulocytic/monocytic differentiation that in some cases caused severe distress after a latency period of more than one year. Intriguingly, upon injection with double transduced NUP98-NSD1 and FLT3-ITD progenitors rather different latency periods of the AML development were observed between different experiments. Interestingly, the latency periods could be correlated to the ratio of expression levels of FLT3-ITD to wildtype FLT3, with higher FLT3-ITD levels associated with a shorter latency. To further investigate the significance of aberrant FLT3 signaling, in vitro and in vivo transformed NUP98-NSD1 and NUP98-NSD1/FLT3-ITD cells were treated with a selective FLT3 tyrosine kinase inhibitor (PKC412). The higher sensitivity of cells co-expressing NUP98-NSD1 and FLT3-ITD to PKC412, compared to cells expressing NUP98-NSD1 only, indicated that proliferation and survival were dependent on FLT3-derived signals.
Taken together, these observations demonstrate a potent cooperation between NUP98-NSD1 fusion and FLT3-ITD in leukemic transformation. However, neither the NUP98-NSD1 fusion protein nor the FLT3-ITD mutation alone was sufficient to induce AML. Moreover, the high sensitivity of NUP98-NSD1 and FLT3-ITD co-expressing leukemic blasts to FLT3 signaling inhibition suggests a possible therapeutic strategy to be further explored in this AML subgroup.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.