Stem cell leukemia/lymphoma syndrome (SCLL) is driven by constitutive activation of chimeric FGFR1 kinases generated by chromosome translocations. We have shown that FGFR inhibitors significantly suppress leukemia and lymphoma development in vivo, and cell viability in vitro. Since resistance to targeted therapies is a major reason for relapse, we developed FGFR1-overexpressing mouse and human cell lines that are resistant to the specific FGFR inhibitors AZD4547 and BGJ398, as well as non-specific inhibitors, such as ponatinib, TKI258 and E3810. Two mutually exclusive mechanisms for resistance were demonstrated; an activating V561M mutation in the FGFR1 kinase domain and mutational inactivation of PTEN resulting in increased PI3K/AKT activity. Ectopic expression of PTEN in the PTEN-mutant cells resensitizes them to FGFR inhibitors. Treatment of resistant cells with BGJ398, in combination with the BEZ235 PI3K inhibitor, shows an additive effect on growth in vitro and prolongs survival in xenograft models in vivo. These studies provide the first direct evidence for both the involvement of the FGFR1 V561M mutation and PTEN inactivation in the development of resistance in leukemias overexpressing chimeric FGFR1. These studies also provide a potential strategy to treat leukemias and lymphomas driven by FGFR1 activation that become resistant to FGFR1 inhibitors.
LTR retrotransposons are repetitive DNA elements comprising ∼10% of the human genome. However, LTR sequences are disproportionately present in human long, non-coding RNAs (lncRNAs). Whether and how the LTR lncRNAs serve biological functions are largely unknown. Here we show that in primary human erythroblasts, lncRNAs transcribed from the LTR retrotransposons of ERV-9 human endogenous retrovirus activated transcription of key erythroid genes and modulated ex vivo erythropoiesis. To dissect the functional mechanism of ERV-9 lncRNAs, we performed genome-wide RNA and ChIRP analyses before and after global knockdown or locus-specific deletion of ERV-9 lncRNAs in human erythroblasts carrying ∼4000 copies of the ERV-9 LTRs and in transgenic mouse erythroblasts carrying a single copy of the primate-specific ERV-9 LTR in the 100 kb human β-globin gene locus. We found that ERV-9 lncRNAs acted in cis to stabilize assembly of the ERV-9 LTR enhancer complex and facilitate long-range LTR enhancer function in activating transcription of downstream, cis-linked globin genes. Our findings suggested that LTR lncRNAs transcribed from many of the 4000 copies of ERV-9 LTR retrotransposons acted by a similar cis mechanism to modulate LTR enhancer function in activating transcription of downstream genes critical to cellular processes including erythropoiesis.
Oncogenic transformation of hematopoietic stem cells by chimeric fusion kinases causing constitutive activation of FGFR1 leads to a stem cell leukemia/lymphoma (SCLL) syndrome, accompanied by widespread dysregulation of gene activity. We now show that FGFR1 activation is associated with upregulation of MYC and pharmacological suppression of FGFR1 activation leads to downregulation of MYC and suppression of MYC target genes. Luciferase reporter assays demonstrate FGFR1 can directly regulate MYC expression and this effect is enhanced in the presence of chimeric FGFR1 kinases. In SCLL cells, a truncated form of FGFR1 is generated by granzyme B cleavage of the chimeric kinases, producing a nucleus-restricted derivative that can bind MYC regulatory regions. Mutation of the granzyme B cleavage site prevents relocation to the nucleus but does not suppress MYC activation, suggesting additional mechanisms of MYC activation in the presence of cytoplasm-restricted chimeric kinases. We show one of these mechanisms involves activating cytoplasmic STAT5, which upregulates MYC independent of the truncated FGFR1 kinase. Targeting MYC function using shRNA knockdown and 10054-F8 in SCLL cells leads to inhibition of cell proliferation and synergizes with the BGJ398 FGFR1 inhibitor, suggesting a combination therapy that could be used in the treatment of SCLL.
MicroRNAs have pathogenic roles in the development of a variety of leukemias. Here we identify miRNAs that have important roles in the development of B-lymphomas resulting from the expression of the chimeric BCR-FGFR1 kinase. The miR-17/92 cluster was particularly implicated and forced expression resulted in increased cell proliferation, while inhibiting its function using microRNA sponges reduced cell growth and induced apoptosis. Cells treated with the potent BGJ389 FGFR1 inhibitor led to miR-17/92 downregulation suggesting regulation by FGFR1. Transient luciferase reporter assays and qRT-PCR detection of endogenous miR-17/92 expression in stable transduced cell lines demonstrated that BCR-FGFR1 can regulate miR-17/92 expression. This positive association of miR-17/92 with BCR-FGFR1 was also confirmed in primary mouse SCLL tissues and primary human CLL samples. miR-17/92 promotes cell proliferation and survival by targeting CDKN1A and PTEN in B-lymphoma cell lines and primary tumors. An inverse correlation in expression levels was seen between miR-17/92 and both CDKN1A and PTEN in two cohorts of CLL patients. Finally, in vivo engraftment studies demonstrated that manipulation of miR-17/92 was sufficient to affect BCR-FGFR1 driven leukemogenesis. Overall, our results define miR-17/92 as a downstream effector of FGFR1 in BCR-FGFR1 driven B cell lymphoblastic leukemia.
Transformation of hematopoietic stem cells by the BCR-FGFR1 fusion kinase found in a variant of Stem Cell Leukemia/Lymphoma (SCLL) syndrome leads to development of B-lymphomas in syngeneic mice and humans. In this study, we show that the relatively rapid onset of this leukemia is potentially related to oncogenic domains within the BCR component. BCR recruited a guanidine nucleotide exchange factor (GEF) domain to the fusion kinase to facilitate activation of small GTPases such as RHOA. Deletion of this GEF domain increased leukemogenesis, enhanced cell survival and proliferation, and promoted stem cell expansion and lymph node metastasis. This suggests that, in an SCLL context, the presence of the endogenous GEF motif leads to reduced leukemogenesis. Indeed, loss of the GEF domain suppressed activation of RHOA and PTEN, leading to increased activation of AKT. Loss of the GEF domain enhanced cell proliferation and invasion potential, which was also observed in cells in which RHOA is knocked down, supported by the observation that overexpression of RHOA leads to reduced viability and invasion. In vivo, depletion of RHOA in SCLL cells significantly increased disease progression and shortened latency. Collectively, these data show that the BCR GEF domain affects phenotypes associated with progression of SCLL through suppression of RHOA signaling.
The development of myeloid and lymphoid neoplasms related to overexpression of FGFR1 kinases as a result of chromosome translocations depends on the promotion of a stem cell phenotype, suppression of terminal differentiation, and resistance to apoptosis. These phenotypes are related to the stem cell leukemia/lymphoma syndrome (SCLL), which arises through the effects of the activated FGFR1 kinase on gene transcription, which includes miRNA dysregulation. In a screen for miRNAs that are directly regulated by FGFR1, and which stimulate cell proliferation and survival, we identified miR-339-5p, which is highly upregulated in cells carrying various different chimeric kinases. Overexpression of miR-339-5p in SCLL cell types enhances cell survival and inhibition of its function leads to reduced cell viability. miR-339-5p overexpression protects cells from the consequences of FGFR1 inactivation, promoting cell-cycle progression and reduced apoptosis. Transient luciferase reporter assays and qRT-PCR detection of endogenous miR-339-5p expression in stably transduced cell lines demonstrated that BCR-FGFR1 can directly regulate miR-339-5p expression. This correlation between miR-339-5p and FGFR1 expression is also seen in primary human B-cell precursor acute lymphoblastic leukemia. In a screen to identify targets of miR-339-5p, we identified and verified the and genes, which can promote apoptosis. , SCLL cells forced to overexpress miR-339-5p show a more rapid onset of disease and poorer survival compared with parental cells expressing endogenous levels of miR-339-5p. Analysis of human primary B-cell precursor ALL shows a significant higher expression of miR339-5p compared with the two cohorts of CLL patient samples, suggesting direct roles in disease progression and supporting the evidence generated in mouse models of SCLL. Proapoptiotic genes that are direct targets of miR-339-5p significantly influence promotion and aggressive development of leukemia/lymphomas associated with FGFR1 overexpression. .
Constitutive activation of FGFR1, as a result of diverse chromosome translocations, is the hallmark of stem cell leukemia/lymphoma syndrome. The BCR‐FGFR1 variant is unique in that the BCR component contributes a serine–threonine kinase (STK) to the N‐terminal end of the chimeric FGFR1 kinase. We have deleted the STK domain and mutated the critical Y177 residue and demonstrate that the transforming activity of these mutated genes is reduced compared to the BCR‐FGFR1 parental kinase. In addition, we demonstrate that deletion of the FGFR1 tyrosine kinase domain abrogates transforming ability, which is not compensated for by BCR STK activity. Unbiased screening for proteins that are inactivated as a result of loss of the BCR STK identified activated S6 kinase and SHP2 kinase. Genetic and pharmacological inhibition of SHP2 function in SCLL cells expressing BCR‐FGFR1 in vitro leads to reduced viability and increased apoptosis. In vivo treatment of SCLL in mice with SHP099 leads to suppression of leukemogenesis, supporting an important role for SHP2 in FGFR1‐driven leukemogenesis. In combination with the BGJ398 FGFR1 inhibitor, cell viability in vitro is further suppressed and acts synergistically with SHP099 in vivo suggesting a potential combined targeted therapy option in this subtype of SCLL disease.
BackgroundIn angiosperm, after the first asymmetric zygotic cell division, the apical and basal daughter cells follow distinct development pathways. Global transcriptome analysis of these two cells is essential in understanding their developmental differences. However, because of the difficulty to isolate the in vivo apical and basal cells of two-celled proembryo from ovule and ovary in higher plants, the transcriptome analysis of them hasn't been reported.ResultsIn this study, we developed a procedure for isolating the in vivo apical and basal cells of the two-celled proembryo from tobacco (Nicotiana tabacum), and then performed a comparative transcriptome analysis of the two cells by suppression subtractive hybridization (SSH) combined with macroarray screening. After sequencing, we identified 797 differentially expressed ESTs corresponding to 299 unigenes. Library sequence analysis successfully identified tobacco homologies of genes involved in embryogenesis and seed development. By quantitative real-time PCR, we validated the differential expression of 40 genes, with 6 transcripts of them specifically expressed in the apical or basal cell. Expression analysis also revealed some transcripts displayed cell specific activation in one of the daughter cells after zygote division. These differential expressions were further validated by in situ hybridization (ISH). Tissue expression pattern analysis also revealed some potential roles of these candidate genes in development.ConclusionsThe results show that some differential or specific transcripts in the apical and basal cells of two-celled proembryo were successfully isolated, and the identification of these transcripts reveals that these two daughter cells possess distinct transcriptional profiles after zygote division. Further functional work on these differentially or specifically expressed genes will promote the elucidation of molecular mechanism controlling early embryogenesis.
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