SRSF2P95Hand FLT3ITDSynergistically Drives Leukemic Transformation in Zebrafish
Author: Lingge Tu, SIN Chun-fung, Yanan Chen, Fangfang He, Dandan Wang, Hoi Yi Chan, Sze Pui Tsui, Alvin Chu Hang Ma, Xuan Sun, Anskar Leung
Internal Tandem Duplication of human FMS-like tyrosine kinase (FLT3-ITD) is one of the most frequent genetic alterations in de novo acute myeloid leukemia (AML) and is often acquired during transformation of myelodysplastic syndrome (MDS) into AML, predicting an unfavorable prognosis. RNA splicing factor mutations (e.g. hot spot mutations in serine and arginine rich splicing factor 2 – SRSF2) are often found in MDS and chronic myelomonocytic leukemia (CMML). However, the clinicopathologic features of co-mutated FLT3-ITD and SRSF2 in MDS/CMML and transformation to AML are presently unclear. Here we investigated whether FLT3-ITD cooperates with SRSF2-P95H to promote leukemogenesis in zebrafish.
Transgenic zebrafish carrying human FLT3-ITD or SRSF2-P95H as driven by Runx1 promoter were generated by Tol2 transgenesis. Tg(Runx1:FLT3ITD) was outcrossed with Tg(Runx1: SRSF2P95H) to generate Tg(Runx1:FLT3ITDSRSF2P95H) zebrafish. The presence of human FLT3-ITD or SRSF2-P95H was confirmed through Sanger sequencing, and the expression of each transcript was detected by qRT-PCR in both embryos and adult kidney marrow. Their clinicopathologic features were examined.
At the embryonic stage, the number of leukocytes (l-plastin+) was significantly increased in Tg(Runx1: FLT3ITDSRSF2P95H) embryos compared with their single mutant and wildtype (WT) siblings, whereas the abundance of neutrophils (SBB+) was unaffected. At six months of age, Tg(Runx1:FLT3ITDSRSF2P95H) zebrafish showed monocytosis in peripheral blood (PB) (1 out of 4), but not in the single mutant and WT siblings. In kidney marrow (KM), there was expansion of both immature and mature monocytes, and dysplasia of granulopoiesis and erythropoiesis. At 9 months of age, all four Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish showed neutropenia and substantial increase in monocytosis. At 1 year of age, one out of the nine Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish showed a significant increase in blasts in KM, reminiscent of human AML, and the other eight Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish demonstrated phenotypes consistent with human CMML-like diseases. Increased monocytic compartment of KM was confirmed by α-naphthyl acetate esterase sensitive to fluoride inhibition. In addition, both the spleen area and cellularity were expanded in Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish compared with Tg(Runx1: SRSF2P95H) single mutant and WT siblings. Taken together, our findings demonstrated that FLT3-ITD cooperates with SRSF2-P95H to drive the initiation and progression of CMML and the subsequent transformation to AML. Understanding the molecular underpinnings of these events will shed light to the diverse clinicopathologic features in CMML patients.