Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes. Here, we find that the RNA-binding protein IGF2BP3, which is overexpressed in MLL-translocated leukemia, strongly amplifies MLL-Af4-mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4-driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. At the molecular level, IGF2BP3 regulates a complex posttranscriptional operon governing leukemia cell survival and proliferation. IGF2BP3-targeted mRNA transcripts include important MLL-Af4-induced genes, such as those in the Hoxa locus, and the Ras signaling pathway. Targeting of transcripts by IGF2BP3 regulates both steady-state mRNA levels and, unexpectedly, pre-mRNA splicing. Together, our findings show that IGF2BP3 represents an attractive therapeutic target in this disease, providing important insights into mechanisms of posttranscriptional regulation in leukemia.
Chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene are observed in acute lymphoblastic leukemias (ALL), acute myeloid leukemias (AML), and in rare mixed-lineage leukemia. Despite recent progress in therapeutic approaches, patients with MLL-rearranged (MLLr) leukemias still have very poor outcomes and a high risk of relapse. Of more than 90 fusion partner genes, MLL-AF4 is the most common MLL fusion protein in patients. Previously, we found that the RNA binding protein IGF2BP3 was specifically overexpressed in MLL-rearranged B-ALL, and enforced expression in vivo led to a pathologic expansion of hematopoietic stem and progenitor cells resulting in B and myeloid cell leukocytosis in the periphery. However, the requirement of IGF2BP3 in MLL-AF4 mediated leukemogenesis remains to be determined. Utilizing our previously generated list of differentially regulated targets with IGF2BP3 knockdown and a published dataset of MLL-Af4 targets, we determined that transcripts modulated by IGF2BP3 showed significant enrichment for MLL-Af4-bound genes. Furthermore, we observed that MLL-AF4 directly binds to and transcriptionally induces IGF2BP3. We performed ChIP-PCR assays on RS4;11 and SEM cell lines, human B-ALL cell lines that carry the MLL-AF4 translocation, and determined that the region in the first intron of IGF2BP3 is strongly bound by MLL-AF4. Furthermore, we observed a dose-dependent increase in luciferase reporter activity when we co-transfected a dual-luciferase reporter vector containing the promoter region of IGF2BP3 with increasing levels of MLL-AF4 expressing retroviral vector. To determine the role of Igf2bp3 in MLL-Af4 driven leukemogenesis, we generated the first Igf2bp3 KO murine model. Surprisingly, Igf2bp3 KO mice maintain normal, steady-state hematopoiesis. However, in striking contrast, deletion of Igf2bp3 in the MLL-Af4 leukemia model, significantly increases the survival of MLL-Af4 transplanted mice and greatly attenuates the disease. Furthermore, Igf2bp3 deficiency significantly reduced the tumor burden and disease severity. We observed significant decreases in WBC counts, spleen weights, and infiltrating leukemic cells visualized in histopathological analysis of hematopoietic tissues and quantified by FACS analysis. Moreover, deletion of Igf2bp3 led to a leukemia-initiating cell (LIC) disadvantage in vivo, demonstrated by significantly reduced engraftment in primary transplanted mice and reconstitution of secondary serially transplanted mice. To identify the transcripts directly regulated by Igf2bp3 in the context of MLL-Af4 driven leukemia, we carried out enhanced crosslinking and immunoprecipitation (eCLIP) transcriptome analysis of MLL-Af4 transformed early stem and progenitor cells and primary cells purified from splenic tumors of MLL-Af4 leukemic mice. We discovered an IGF2BP3-regulated post-transcriptional operon governing leukemic cell survival and proliferation, in which mRNA targets include the Hoxa locus and numerous genes within the Ras signaling pathway. In our study, we provide evidence that Igf2bp3 is required for the initiation of MLL-Af4 driven leukemia. We determined that Igf2bp3 is necessary for the development of and function of MLL-Af4 LICs. Mechanistically, we show that Igf2bp3 binds to and modulates the expression of hundreds of critical target transcripts. In summary, we demonstrate that Igf2bp3 is a positive regulator of MLLr leukemogenesis by targeting Hoxa transcripts such as Hoxa9 and numerous Ras signaling pathway transcripts, thereby controlling multiple downstream effector pathways required for disease initiation and aggressiveness. Together, our findings identify IGF2BP3 as an important, potential therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare.
Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes and a high risk of relapse. Here, we found that MLL-AF4, the most common MLL fusion protein in patients, transcriptionally induces IGF2BP3 and that IGF2BP3 strongly amplifies MLL-Af4 mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4 driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. eCLIP and transcriptome analysis of MLL-Af4 transformed stem and progenitor cells and MLL-Af4 bulk leukemia cells reveals a complex IGF2BP3-regulated post-transcriptional operon governing leukemia cell survival and proliferation. Regulated mRNA targets include important leukemogenic genes such as those in the Hoxa locus and numerous genes within the Ras signaling pathway. Together, our findings show that IGF2BP3 is an essential positive regulator of MLL-AF4 mediated leukemogenesis and represents an attractive therapeutic target in this disease.
Background Intraoperative neurophysiological monitoring (IONM) is utilized for both the localization of critical structures and for real time detection and prevention of intraoperative neurological injury. Use of IONM to monitor the hypoglossal nerve is performed during neurosurgical, otolaryngological, and vascular procedures to improve surgical outcomes. There is a paucity of literature describing potential complications of IONM of the hypoglossal nerve, especially with respect to airway compromise. Here we present our findings regarding a case of acute airway obstruction following hypoglossal nerve monitoring. Case Presentation A 54-year-old male was admitted for left far-lateral craniotomy and microsurgical clipping of a left posterior inferior cerebellar artery (PICA) aneurysm. Following induction and intubation but prior to the procedure start, the patient was placed in the ¾ prone position with the left side up and his neck was flexed approximately 10 degrees. He then underwent placement of subdermal needle electrodes into the facial muscles, trapezius muscles, soft palate, and tongue for IONM. The procedure lasted 523 minutes and was completed without complication. However, approximately one hour after emergence from general anesthesia, the patient experienced progressive difficulty breathing secondary to severe lingual swelling. He required emergent placement of a nasotracheal tube guided by a fiberoptic bronchoscope. He remained intubated for 3 days and was treated with dexamethasone, after which the swelling resolved, and the patient was successfully extubated. Conclusions Acute lingual edema is a potentially life-threatening phenomenon that can lead to rapid airway compromise. Generally, causes of acute lingual swelling include hemorrhage, edema, infarction, and infection. In the case described above, we suspect traumatic injury to the tongue’s vascular supply caused a deep tissue hematoma leading to postoperative acute lingual swelling and airway obstruction. With the widespread use of IONM, it becomes essential for providers to be aware that perioperative airway compromise is a potentially life-threatening complication, especially with respect to monitoring of the hypoglossal nerve. Awake fiberoptic nasotracheal intubation can successfully be employed to establish an emergency airway in such situations.
Acute lymphoblastic leukaemia (ALL) is the most common pediatric haematological malignancy with a prevalence of around 85% cases of B cell origin. ETV6-RUNX1 is the most common genetic aberration in childhood B-ALL with a prevalence of around 25% in Western countries and a lower prevalence in Asian countries. ETV6-RUNX1 presence portends a good prognosis in B-ALL. ETV6-RUNX1 translocation has been observed in 1-5% of healthy newborns, indicating that it is not sufficient for leukemogenesis, and the need to accumulate secondary mutations for leukemogenesis. We have previously demonstrated the specific overexpression of Insulin like Growth Factor 2 Binding Protein 1 (IGF2BP1), an oncofetal RNA binding protein, in ETV6-RUNX1 positive B-ALL. Overexpression of the ETV6-RUNX1 fusion protein in 7OZ/3, a murine B-ALL cell line increased endogenous Igf2bp1 levels in a dose dependent manner. Conversely, knockout of IGF2BP1 in Reh-Cas9-GFP expressing B-ALL cell line, a modified version of Reh (cell line positive for ETV6-RUNX1 translocation), using 2 different guide RNAs led to a decrease in the ETV6-RUNX1 transcript expression in the IGF2BP1 KO Reh-Cas9 cells. This was accompanied by a decrease in cell proliferation and increased chemosensitivity to prednisolone. A similar phenotype was observed in Reh-Cas9 cells after knockout of ETV6-RUNX1 fusion transcript hinting towards a synergism between the two proteins in tumour cell survival and chemosensitivity. Interestingly, there appears to be a positive correlation between prednisolone resistance and ETV6-RUNX1 positivity in our patient cohort too, thus supporting the in vitro finding. Previous studies have shown the binding of IGF2BP1 to the ETV6-RUNX1 fusion transcript which was also confirmed by us. To study the in vivo synergistic effect between ETV6-RUNX1 fusion protein and IGF2BP1, we used a murine bone marrow transplant model. The individual genes were overexpressed alone (IGF2BP1/ETV6-RUNX1) or together (IGF2BP1+ETV6-RUNX1) in separate groups of lethally irradiated mice (n=8 per group). In the group where both the genes were overexpressed together, there was clonal expansion and hypercellularity in the bone marrow. This was accompanied by an increase in the number of lineage negative cells (Lin-), progenitors including HSCs, LMPPs and c-kit+ cells which also showed high Ki67 positivity. The marrow hypercellularity led to extramedullary haematopoiesis which was seen as significant splenomegaly. In addition, analysis of the endogenous levels of Igf2bp1 in the bone marrow of the primary murine samples belonging to the ETV6-RUNX1 fusion protein overexpression group resulted in an increase in its levels, thus confirming our in vitro finding suggesting its induction by ETV6-RUNX1 fusion protein. RNA-Seq of IGF2BP1 KO cells identified enrichment of targets of the TNF alpha/NFκB signalling and K-Ras pathways hinting towards their dependency on IGF2BP1. The direct targets of IGF2BP1 were identified by RIP-Seq (RNA-Immunoprecipitation-Seq) analysis in Reh cell line. Numerous genes from the oncogenic signalling pathways including the TNF alpha/ NFκB signalling pathway were identified as IGF2BP1 targets. We validated some overexpressed genes from the TNF alpha/NFκB signalling and PI3K pathway in primary patient samples. We found significant overexpression of some of the genes (IL6ST, NFAT5, CDK6, MDM2, CCND1, NGFR) belonging to these pathways in the ETV6-RUNX1 positive group (n=39) compared to the negative cohort (n=111) as well as MACS sorted CD19+ B-cells (n=5). The decrease in the ETV6-RUNX1 transcript levels after downregulation of IGF2BP1 and overlap between the oncogenic pathways clearly shows an interdependency between the two genes. Overall, our results suggest a potential feedback mechanism between ETV6-RUNX1 fusion protein and IGF2BP1 in the pathogenesis of leukemia development and IGF2BP1 can be utilised as an ideal therapeutic target for this particular subtype. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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