RHOA-regulated IGFBP2 promotes invasion and drives progression of BCR-ABL1 chronic myeloid leukemia.

The Philadelphia 9;22 chromosome translocation has two common isoforms that are preferentially associated with distinct subtypes of leukemia. The p210 variant is the hallmark of chronic myeloid leukemia (CML) whereas p190 is frequently associated with B-cell acute lymphoblastic leukemia. The only sequence difference between the two isoforms is the guanidine exchange factor domain. This guanidine exchange factor is reported to activate RHO family GTPases in response to diverse extracellular stimuli. It is not clear whether and, if so, how RHOA contributes to progression of p210 CML. Here we show that knockout of RHOA in the K562 and KU812, p210-expressing cell lines leads to suppression of leukemogenesis in animal models in vivo. RNA-sequencing analysis of the mock control and null cells demonstrated a distinct change in the gene expression profile as a result of RHOA deletion, with significant downregulation of genes involved in cell activation and cell adhesion. Cellular analysis revealed that RHOA knockout leads to impaired cell adhesion and migration and, most importantly, the homing ability of leukemia cells to the bone marrow, which may be responsible for the attenuated leukemia progression. We also identified IGFBP2 as an important downstream target of RHOA. Further mechanistic investigation showed that RHOA activation leads to relocation of the serum response factor (SRF) into the nucleus, where it directly activates IGFBP2. Knockout of IGFBP2 in CML cells suppressed cell adhesion/invasion, as well as leukemogenesis in vivo. This elevated IGFBP2 expression was confirmed in primary CML samples. Thus, we demonstrate one mechanism whereby the RHOA-SRF-IGFBP2 signaling axis contributes to the development of leukemia in cells expressing the p210 BCR-ABL1 fusion kinase.

A truncated derivative of FGFR1 kinase cooperates with FLT3 and KIT to transform hematopoietic stem cells in syndromic and de novo AML.

BACKGROUND: Myeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated as a result of chromosome translocations, which lead to acquisition of dimerization motifs in the chimeric proteins. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated. METHODS: Stem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq was used to examine the related signaling pathways and regulated target genes. RESULTS: For the first time, we show that this non-classical truncated form of FGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells in an animal model in vivo. These leukemia cells show a mixed immunophenotype with a B-cell B220 + Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. tnFGFR1, however, does not activate classic FGFR1 downstream signaling pathways but induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. We further show that de novo human AML also express tnFGFR1 which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but treatment of these cells with the Quizartinib (AC220) FLT3 inhibitor, suppresses in vitro growth and development of leukemia in vivo. Combined treatment with FGFR1 and FLT3 inhibitors provides increased survival compared to FGFR1 inhibition alone. CONCLUSIONS: This study demonstrates a novel model for transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by the truncated nuclear tnFGFR1 derivative, which is associated with GZMB expression levels. Genes significantly upregulated by tnFGFR1 include Flt3 and Kit which promote a leukemia stem cell phenotype. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with an inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile.