Cooperativity of IGF2BP1 and ETV6-RUNX1 in the pathogenesis of ETV6- RUNX1 positive B-Cell Acute Lymphoblastic Leukemia

DOI: https://doi.org/10.21203/rs.3.rs-1691465/v1

Abstract

Background

ETV6-RUNX1 is the most prevalent translocation in pediatric B-ALL (B-cell Acute Lymphoblastic Leukemia). However, the exact pathogenesis of this translocation leading to leukemogenesis remains unclear. Insulin like Growth Factor 2 Binding Protein 1 (IGF2BP1), an oncofetal RNA binding protein (RBP), is overexpressed in ETV6-RUNX1 positive B-ALL. We intended to investigate the role of IGF2BP1 in leukemogenesis in this study.

Methods

qRT-PCR was used to analyze IGF2BP1 overexpression in an Indian cohort of pediatric B-ALL (n = 167). IGF2BP1 and ETV6-RUNX1 were knocked out in Reh, an ETV6-RUNX1 positive B-ALL cell line, using CRISPR/Cas9 technology. Cell proliferation assays were used to study cell survival after IGF2BP1/ETV6-RUNX1 KO and prednisolone response. RNA-Sequencing after IGF2BP1 knockout and RNA Immunoprecipitation sequencing (RIP-Seq) after IGF2BP1 pulldown were performed to find the putative targets of IGF2BP1. Luciferase reporter assays were used to study downstream mRNA target stability as well as pathways regulated by IGF2BP1. Finally, to study the in-vivo synergistic effect between ETV6-RUNX1 fusion protein and IGF2BP1, a murine bone marrow transplant model was utilized followed by flowcytometric analysis of hematopoietic tissues and histopathological examination.

Results

IGF2BP1 expression was found to be essential for tumor cell survival in Reh cell line. Knockout of IGF2BP1 or ETV6-RUNX1 led to reduced proliferation and increased sensitivity to prednisolone hinting at synergism between both the genes. Integrated analysis of transcriptome sequencing after IGF2BP1 knockout and RIP-Seq revealed that IGF2BP1 targets encompass multiple pro-oncogenic signalling pathways including TNFα/NFκB and PI3K-Akt pathways. Dysregulation of these pathways was also observed in patient samples. Mechanistically, we demonstrate that IGF2BP1 binds and stabilizes ETV6-RUNX1 fusion transcript and the presence of a positive feedback loop between them which maintains a constitutive dysregulation of several oncogenic pathways.

Background

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy with ~ 85% being of B-cell origin (B-ALL) [1]. B-ALL is characterized by the presence of different translocations including BCR-ABL, ETV6-RUNX1, E2A-PBX1, MLL fusion rearrangement, and several novel subtypes classified by gene expression signatures [2]. ETV6-RUNX1 is the most common translocation with an incidence of ~ 25% in Western countries and a lower incidence in India and its neighboring countries [3, 4]. The ETV6-RUNX1 fusion occurs as an early, prenatal event in-utero and results in the formation of a pre-leukemic clone [5] which converts to ALL at a later stage after acquiring secondary mutations [6]. Despite its association with a good prognosis, some ETV6-RUNX1 patients have an overall poorer prognosis, usually associated with relapse [7].

ETV6-RUNX1 is a weak oncogene and its enforced expression in mouse hematopoietic stem cells (HSCs)/committed progenitors does not result in the development of B-ALL. Leukemia development was observed at a low penetrance when combined with loss of one of the alleles of Kdm5c/Pax5/Cdkn2a or when exposed to infections [8–10].

IGF2BP1, an oncofetal RNA binding protein (RBP), is known to be overexpressed in several cancers [11] including B-ALL [12]. IGF2BP1 has been seen to promote leukemic stem cell (LSC) maintenance and survival [13]. A GWAS study identified IGF2BP1 expression to be unique in ETV6-RUNX1 translocation positive B-ALL patients [14]. ETV6-RUNX1 transcript is a known target of IGF2BP1 [12]. Overexpression of IGF2BP1 in the bone marrow has been found to be highly specific for diagnosing the presence of the ETV6-RUNX1 translocation [15].

In this study, we extend our current knowledge in understanding the role of IGF2BP1 overexpression in expediting the pathogenesis of ETV6-RUNX1 positive B-ALL. We demonstrate IGF2BP1 overexpression in ETV6-RUNX1 B-ALL patient samples and also show the presence of prednisolone resistance and relapses in a subset of these patients. CRISPR mediated knockout of IGF2BP1 or ETV6-RUNX1 led to reduced tumor cell survival and reversal of prednisolone resistance. The transcriptome regulated by IGF2BP1 included numerous pro-oncogenic signalling pathways including the TNF alpha/NFκB and the PI3K-Akt pathways. We validated the upregulation of some of these putative IGF2BP1 target genes in a B-ALL patient cohort.

A bone marrow transplant model co-expressing ETV6-RUNX1 and IGF2BP1 in lethally irradiated mice led to the development of a pre-leukemic phenotype that included clonal expansion in the bone marrow which was primarily led by uncommitted, progenitor proliferation. Overall, our results suggest that IGF2BP1 plays an important role in the pathogenesis of ETV6-RUNX1 leukemia through multiple oncogenic pathways and can be utilized as an ideal therapeutic target for this particular subtype.

Methods

Patient sample collection and processing

Treatment naïve B-ALL patient bone marrow (BM) samples were collected from March 2016 to December 2020 at BR Ambedkar Institute Rotary Cancer Hospital at AIIMS, New Delhi. The study was approved by the Institutional Ethics and Biosafety Committees (IEC-1950/1.04.2016, RP-20/2016). Samples were collected after informed consent from a guardian and assent was taken from children > 7 years of age in accordance with the Declaration of Helsinki regulations. In addition, some archival BM samples, preserved from previous studies, were also utilized after proper ethics clearance. Peripheral blood from healthy controls was also collected.

Cell Culture

The human cell lines HEK 293T (ATCC® CRL3216™), Reh (ATCC CRL-8286), murine pre-B-ALL cell line 7OZ/3 (ATCC® TIB-158™) were obtained from American Type Culture Collection (ATCC) and maintained as described previously [16].

Rna Isolation And Real-time Pcr

Total RNA was extracted from BM/cell lines using the TRIzol (Takara) method. 500-1000ng of RNA was reverse transcribed to cDNA using MMLV RT (Thermo Scientific, USA). Real Time PCR was performed to quantify gene expression using TB Green Premix Ex II (Takara). RNA Polymerase II and PPIA were used as internal controls for mammalian cell lines and mGapdh and mL32 for murine cell lines. ΔΔCt method was used to compare the gene expression [17].

Crispr/cas9 Ko

Reh-Cas9 was created by transducing Reh with LentiCas9-GFP overexpressing lentiviruses. Guide RNAs were designed using the Zhang lab website (http://crispr.mit.edu/) and cloned into pLKO5-EFS-tRFP vector [18]. Lentiviruses and retroviruses were generated as previously described [19].

Statistical Analysis

The patients were followed up in the Medical Oncology department, BRAIRCH, AIIMS. The last follow up was upto December 31, 2021. Overall survival (OS) was calculated as the time duration from date of diagnosis to death or last follow-up. Event free survival (EFS) was calculated as the time duration from date of diagnosis to the date of last follow-up or the first event (relapse or death). The probability of EFS and OS was calculated by the Kaplan-Meier method, with the differences compared using a two-sided log-rank test. All in-vitro experiments were repeated at least thrice. Mouse experiments were done at least twice. Comparison between different groups was done using Mann Whitney (two groups) /Kruskal-Wallis (more than two groups) statistical tests wherever applicable using GraphPad Prism software version 5 or SPSS statistical software package v20. A p-value of < 0.05 was considered to be significant. Additional methods and reagent details are provided in the Supplementary Methods and Tables 1–5.

Results

IGF2BP1 expression is specific to ETV6-RUNX1 positive patient samples

We had earlier reported the overexpression of IGF2BP1 by RT-qPCR in the bone marrow of patients belonging to the ETV6-RUNX1 subtype in a cohort of Indian patients (n=114) [20]. Additional samples were added to the same cohort (Total n=167). IGF2BP1 expression was found to be significantly higher (>1000 fold) in ETV6-RUNX1 subtype, (median value = 2.34) compared to ETV6-RUNX1 negative samples (median value = 0.0022) (p <0.0001) (Fig. 1A).

The presence of the ETV6-RUNX1 translocation also correlated with lower White Blood Corpuscle (WBC) counts (Fig. 1B). For analyzing prednisolone response, the samples were stratified into ETV6-RUNX1 positive and negative groups. The negative group included patients from the other translocations group (BCR-ABL1, E2A-PBX1 and MLL) and the No Known Sentinel Translocation (NKST) group. We observed a higher proportion of Prednisolone Poor Responders (PPRs) among the ETV6-RUNX1 positive patients (38% vs 26%) which was statistically non-significant (Fig. 1C).

The presence of this translocation correlated with a better overall survival as well as lower percentage of deaths (p=0.034, NKST vs E6R1+ and p<0.0001, E6R1- vs E6R1+) (median survival 39 months for NKST and 21 months for E6R1- patients and >60 months for E6R1+ patients) (Fig. 1D) which corroborates with existing literature [21]. Despite showing a better event free survival (p=0.017, NKST vs E6R1+ and p<0.0001, E6R1- vs E6R1+) (median survival was 56 months for E6R1+ patients compared to 33 months for NKST and 19 months for E6R1- patients), around 23% of the ETV6-RUNX1 positive patients relapsed (Fig. 1E).

IGF2BP1 loss of function inhibits B-ALL cell proliferation and reverses prednisolone resistance

IGF2BP1 expression was observed to be significantly higher in Reh (ETV6-RUNX1 translocated) cell line when compared to other leukemic cell lines (Fig. 2A). To examine the effects of the loss of IGF2BP1, knockout was performed in Reh-Cas9 cell line using 3 different sgRNAs (sg1-3) which were designed and cloned as described previously [18] (Fig. 2B). Knockout was confirmed by Western Blotting with sg1 and sg2 causing complete knockout (Fig. 2C). IGF2BP1 knockout led to a significant decrease in cell proliferation as seen by the MTS assay.

IGF2BP1 knockout reversed prednisolone resistance and cells showed sensitivity to prednisolone with IC50 ~1 μM for both the guides used (Fig. 2D-E). Cells with the non-targeting gRNA (NT) were viable even at a 10 μM concentration of prednisolone, confirming resistance [22].

Integrated transcriptome and immunoprecipitation sequencing reveals overlap between IGF2BP1 and ETV6-RUNX1 regulated oncogenic pathways

To begin to unravel the mechanistic basis of our observations, the gene expression profile of IGF2BP1 KO Reh cells was analysed by RNA-Seq. 88 transcripts showed upregulation and 270 transcripts showed downregulation (at least 2-fold change, p<0.05) after IGF2BP1 knockout. Gene Set Enrichment Analysis (GSEA) [23] revealed a significant negative enrichment of the TNFα signalling via NFκB, hypoxia and cholesterol metabolism pathways (Fig. 2E) in KO cells as compared to non-targeting controls. A significant positive enrichment was found for pathways regulating cell cycle and proliferation including G2M checkpoint and the MYC targets (Fig. 2F).

To gain insights into the mRNA interactome of IGF2BP1, we performed RNA Immunoprecipitation followed by high throughput sequencing (RIP-Seq) (Fig. 3A). Application of SETEN [24], a GSEA-based tool for RNA-binding proteins, revealed enrichment of putative IGF2BP1 targets in numerous pro-oncogenic signaling pathways. The ENRICHR tool [25] classified IGF2BP1 target transcripts to be involved in B-cell and myeloid malignancies using the GWAS catalogue database. GO biological processes and molecular functions revealed enrichment for RNA processing, translation and RNA degradation pathways (Fig. 3B, Supplementary Fig.S1A-D).

An integrated analysis of the IGF2BP1-KO RNA-Seq and IGF2BP1 RIP-Seq in Reh cells was performed. Initially, we did not observe a significant enrichment between IGF2BP1 putative targets (RIP-Seq log2 fold>0, Fig. 3C) and the direction of regulation after IGF2BP1 deletion. However, integrating both datasets using GSEA (Fig. 3D) showed that the genes most downregulated after IGF2BP1 deletion were preferentially classified as IGF2BP1 putative targets (highest RIP-Seq enrichment, familywise error-rate p-value<0.003, Fig. 3D). This suggests that IGF2BP1 promotes RNA stability in Reh cells. Interestingly, genes up-regulated after IGF2BP1 KO were preferentially not enriched in the RIP data (familywise error-rate p-value<0.001, Fig. 3D) suggesting that these genes are indirectly regulated by IGF2BP1 (Fig.3C-D).

Combined pathway analysis of IGF2BP1 KO/RIP gene sets revealed that some of the significantly dysregulated pathways after the knockout also had many genes enriched in the RIP-Seq suggesting direct binding and regulation (Fig.3E, Supplementary Data). Examples include the TNFα/NFκB signaling and Hypoxia pathways. Oxidative phosphorylation was upregulated after knockout while cholesterol metabolism was downregulated. However, both were not RIP targets implying an indirect or downstream effect of IGF2BP1 knockout on those pathways. A deeper analysis of genes within these pathways showed that some of the genes in the metabolism of RNA, oxidative phosphorylation, MTORC1 and MAPK pathways showed enrichment in the RIP dataset (Fig.3F).

TNFα induced NFκB and PI3K-Akt signalling pathways are activated in ETV6-RUNX1 positive B-ALL by IGF2BP1

We then aimed to map IGF2BP1-induced gene expression changes to expression signatures of different patient subtypes. We applied GSEA to compare our IGF2BP1-KO data to differential gene expression between ETV6-RUNX1, E2A-PBX1 and MLL positive patients (from GSE65647, n=44) [16]. We also analyzed a dataset which compared ETV6-RUNX1 positive patients’ gene expression (n=4) with normal CD19 positive B-cells (n=2) [26]. There was a significant overlap between genes overexpressed and pathways enriched in ETV6-RUNX1 positive tumors and genes/pathways downregulated after deletion of IGF2BP1 (Fig.3G, Supplementary Fig.S2-S4).

We used BTYNB, a functional inhibitor of IGF2BP1 [27] to validate its role in regulating the non-canonical TNFα/NFκB pathway. Treatment of Reh with BTYNB led to a dose dependent decrease in cell proliferation. BTYNB treatment led to a decrease in both IGF2BP1 and ETV6-RUNX1 fusion transcript expression by qPCR. BTYNB had no effect on the RL and Jurkat cell lines which express minimal levels of IGF2BP1(Supplementary Fig. S5A-D).

Reh cells were transduced with NFκB-Luc-dTomato plasmid [28] which consists of the NFκB consensus sequence upstream of the Luciferase reporter gene. These cells showed an increase in luminescence after treatment with TNFα. Pre-treatment of these cells with BTYNB led to a loss of this luminescence induction in a dose dependent manner (Fig.4A-B).

We further validated genes from the TNFα/NFκB and PI3K-Akt pathways in our patient samples along with MACS enriched CD19+ B-cells as controls (Fig. 4C-D). Many of these genes including IL6ST, MDM2, CDK6 and NGFR showed significant upregulation in the ETV6-RUNX1 positive patients.

ETV6-RUNX1 fusion transcript is stabilized by IGF2BP1

ETV6-RUNX1 fusion transcript has been shown to affect the functions of wild type ETV6 as well as RUNX1 in a dominant negative fashion [29]. We sub cloned the ETV6-RUNX1 transcript from a pcDNA3.1-ETV6-RUNX1 plasmid (a kind gift from Dr Anthony Ford, ICR, London) into a bicistronic, retroviral, murine overexpression vector (Supplementary Fig. S6A). ETV6-RUNX1 virus particles were produced as described previously [19] and used to overexpress the same in a murine pre-B-ALL cell line, 7OZ/3 in three serial dilutions. There was a dose dependent increase in Igf2bp1 levels (Fig. 5B).

We performed ETV6-RUNX1 KO using guides targeting the ETV6-RUNX1 junction or the 5’ ETV6 region as reported previously (Supplementary Fig. S6B) [30] in Reh cell line. We observed a decrease in cell proliferation and sensitivity to prednisolone similar to the phenotype seen with IGF2BP1 KO (IC50=1.6/2/3.5 μM for sg1/2/3 respectively). This suggested the involvement of both the proteins in conferring a glucocorticoid-resistant phenotype to Reh cells. (Fig. 5B-C).

IGF2BP1-RIP followed by RT-PCR of immunoprecipitated RNA showed significant enrichment of the ETV6-RUNX1 junction, 5’-UTR of ETV6 and ACTB (positive control) in the RIP vs Input samples (Fig. 5D). In order to study the effect of IGF2BP1 on ETV6-RUNX1 stability, we cloned the junction region downstream of firefly luciferase (Supplementary Fig. S6C). Cotransfection of this vector with IGF2BP1 resulted in significantly increased luciferase activity (Fig. 5E). Complete knockout of IGF2BP1 (sg1 and sg2) significantly decreased ETV6-RUNX1 transcript levels in Reh cells (Fig. 5F). This data demonstrated the regulation of IGF2BP1 by ETV6-RUNX1 and its stabilization by the former.

IGF2BP1 and ETV6-RUNX1 synergize to cause clonal progenitor expansion in the murine bone marrow

To directly assess the synergism between ETV6-RUNX1 and IGF2BP1, we undertook an in-vivo experiment to examine the effects of enforced expression of both transgenes. Murine bone marrow transplant experiments were performed using retroviruses, synthesized and used as previously described [19] (SupplementaryFig. S7A). We cloned the human coding sequence of IGF2BP1 into MIG (MSCV-IRES-GFP), a murine stem cell virus–based (MSCV) retroviral vector and used it along with the ETV6-RUNX1 cloned in MICH (MSCV-IRES-mCherry). We confirmed the functionality of the vectors in expressing IGF2BP1 and ETV6-RUNX1 as well as the fluorescent markers (Supplementary Fig. S7B–D and data not shown).

All groups had similar levels of engraftment as seen from the CD45.1 positivity (data not shown). Clonal expansion was observed in the peripheral bleeds of mice expressing both ETV6-RUNX1 and IGF2BP1 (combination group) from weeks 4-16 with a constant increase. This was measured by the ratio of GFP+ mCherry+ cells to the total transfected cells (double positive ratio) (Supplementary Fig. S8A-B).

Complete blood counts (CBC) at week 16 showed a decrease in mature B-cell counts after ETV6-RUNX1 overexpression and in the combination. There was an increase in total WBC counts after IGF2BP1 overexpression. In the combination, the number of mature red blood cells, platelets and neutrophils were significantly lower with an increase in immature reticulocyte counts (SupplementaryFig. S9A-H).

To further characterize these hematopoietic changes, mice were euthanized after 16 weeks and hematopoietic organs collected for analysis. The bone marrow counts were significantly higher in the combination group with significant clonal expansion. Analysis of the progenitors revealed significant increase in the Lin- population, hematopoietic stem cells (HSCs), Lymphomyeloid Primed Progenitors (LMPP) and common lymphoid progenitors (CLPs) (percentages and absolute counts) in the combination group. The ETV6-RUNX1 group also showed a small but significant increase in progenitor output (Fig. 6A-6G). Hardy fraction analysis of the bone marrow in the combination showed an accumulation of the pre-B cell population implying a B-cell developmental block. (Supplementary Fig.S10).

Previous studies have identified the Lin- c-Kit+ or the LSK (Lin- c-Kit+Sca1+) populations as leukemia initiating cells in different mouse models [31]. The bone marrow of the combination group showed a significant increase in both populations (Fig. 7A) with strong Ki67 positivity of the Lin- progenitor population implying an increased proliferation rate (Fig. 7B). The myeloid progenitors (common myeloid progenitors (CMP), granulocyte-monocyte progenitor (GMP) and megakaryocyte-erythroid progenitor (MEP)) were also significantly increased in the combination (Supplementary Fig.S11).

The progenitor populations from the bone marrow were analyzed for fluorescent marker expression (double positive (DP): mCherry+ GFP+ and double negative (DN): mCherry-GFP-). In the combination group, there was an increase in the Lin- population in both the DN and DP fractions implying both a cell extrinsic and intrinsic mechanism for progenitor expansion. Interestingly, the HSCs and LMPPs were only increased in the DP fraction indicating a purely cell intrinsic mechanism (Supplementary Fig.S12).

Histopathological examination of the bone marrow of mice expressing both ETV6-RUNX1 and IGF2BP1 showed marked hypercellularity along with a loss of normal architecture characterized by compressed vascular spaces, reduced fat globules and megakaryocytes (Fig. 7C). Spleens were significantly enlarged with clonal expansion (Supplementary Fig.S13A-C). The spleen was populated by immature cells including HSCs implying extramedullary hematopoiesis (Supplementary Fig.S13D). Histopathological analysis revealed loss of architecture with red pulp expansion and smaller germinal centers in the combination group (Supplementary Fig.S13E).

RT-qPCR from the bone marrow of the mice showed a significant increase in endogenous Igf2bp1 levels after ETV6-RUNX1 overexpression, validating our in-vitro finding. The levels of ETV6-RUNX1 and IGF2BP1 were highest in the combination group (Fig. 7D).

Discussion

The molecular mechanism of ETV6-RUNX1 mediated leukemogenesis is incompletely understood. ETV6-RUNX1 is known to be a weak oncogene unable to induce leukemia in the absence of secondary genetic alterations [9, 32]. As detected from the bloodspots in newborns, this translocation shows a far higher prevalence, with very few going on to develop leukemia supporting the ‘two-hit’ model of its molecular pathogenesis [33]. Cdkn2a loss, alterations of Epor, Ebf1, Jak1, Jak3, Il2rb, Stat5 and Trp53 have all been shown to synergize with ETV6-RUNX1 to cause leukemia in mice [32, 34]. We had previously identified that the RBP IGF2BP1 was specifically overexpressed in the ETV6-RUNX1 translocated B-ALL and validated the same in a larger cohort [20]. The importance of RBPs in the pathogenesis of various leukemias including the IGF2BP family is slowly being dissected [35]. Our findings add to this existing knowledge of dysregulated RBP expression in ETV6-RUNX1 translocated B-ALL.

B-ALL treatment at our cancer center is done using the Indian Childhood Collaborative Leukemia Group (ICiCLe) Protocol [36]. One of the risk stratification parameters includes percentage of blasts on day 8 after treatment with high dose prednisolone which divides patients into good and poor responders (PGR/PPR) [37]. Glucocorticoid resistance has been linked to poor prognosis and decreased event free survival [38]. The presence of ETV6-RUNX1 translocation appears to correlate with a trend of prednisolone resistance. Although, the presence of ETV6-RUNX1 correlated with a good prognosis as previously reported [39], the event free survival was inferior in comparison to the overall survival. These findings highlight the varied nature of response to chemotherapy and poorer prognosis in at least a subset of the ETV6-RUNX1 positive patients.

Previously, loss of function of IGF2BP1 in epithelial cell lines has been shown to decrease cell proliferation and cause apoptosis [13, 40]. We found that knockout of IGF2BP1 led to reduced cell proliferation and reversal of prednisolone resistance in Reh, an ETV6-RUNX1 positive cell line. A similar phenotype was also observed after ETV6-RUNX1 knockout. Interestingly, some of the pathways upregulated after IGF2BP1 knockout are known to be upregulated after ETV6-RUNX1 knockdown [41]. Knockout of IGF2BP1 also led to a decrease in the expression of ETV6-RUNX1 transcript and overexpression of ETV6-RUNX1 lead to a dose dependent increase in IGF2BP1 levels implying interdependency between the two genes. Previous studies have also identified ETV6-RUNX1 to be a target of IGF2BP1 and a 17q21 polymorphism within IGF2BP1 to be having a strong association with ETV6-RUNX1 positive B-ALL [14, 42].

A mechanistic examination of the transcriptome controlled by IGF2BP1 was analysed via a combined interrogation of the RIP-Seq/IGF2BP1 KO RNA-Seq datasets which revealed enrichment of numerous pro-oncogenic pathways. Many pathways regulated by IGF2BP1 were also significantly associated with ETV6-RUNX1 positive tumors, implying some degree of cooperativity between the two genes in disease pathogenesis.

IGF2BP1 appeared to bind and stabilize genes in the TNFα mediated non-canonical NFκB pathway which was also validated in our patient cohort. The non-canonical NFκB pathway is known to play an important role in inflammation [43]. In ETV6-RUNX1 translocated tumors, it may be contributing to creating an inflammatory environment within the bone marrow niche for the maintenance or emergence of the leukemic clones as reported previously [44]. An in-vitro NFκB mediated luciferase reporter assay showed a decrease in luciferase activity after IGF2BP1 inhibition underscoring the clear role of IGF2BP1 in regulating this pathway.

Interestingly, the pathways which were negatively enriched in the IGF2BP1 KO cells were all positively enriched in the ETV6-RUNX1 positive tumors and vice versa. A recent study has demonstrated that ETV6-RUNX1 functions by competing for RUNX1 binding sites and leads to transcriptional repression of RUNX1 targets. The study also showed ETV6-RUNX1 knockdown leading to an increase in the G2M checkpoint, E2F and MYC target pathways. These pathways were also upregulated after IGF2BP1 knockout further establishing a role for their synergism [45].

RIP-Seq identified numerous pro-oncogenic pathways including the PI3K-Akt and MAPK pathways. Interestingly, some targets identified in RIP-Seq were not significantly downregulated after IGF2BP1 knockout implying a multifactorial regulation of their mRNA stability. A qPCR validation of PI3K-Akt pathway genes (MDM2, CDK6, CCND1, NGFR) demonstrated a significantly high expression in ETV6-RUNX1 positive patients. ETV6-RUNX1 is known to increase MDM2 which normally degrades p53 [46]. Interestingly, MDM2 is also known to have a p53-independent role in childhood ALL where its elevated expression induces expression of p65 subunit of NFκB and augments chemoresistance [47]. We had previously demonstrated CDK6 to be a target of IGF2BP3 in ALL suggesting some degree of overlap between targets of this family [16]. Cyclin D1 (CCND1) is required during the transition from G1 to S phase and is known to be overexpressed in B-cell lymphomas [48]. CCND1 expression is associated with poor prognosis and relapse in childhood ALL [49]. High NGFR expression has been known to be associated with ETV6-RUNX1 rearrangement [50].

To study the pathogenetic role of IGF2BP1 in leukemia development, we developed the first in-vivo model with enforced expression of IGF2BP1 in the hematopoietic system. The combination of IGF2BP1 and ETV6-RUNX1 led to a hypercellular bone marrow along with increased progenitor output across all lineages (HSCs, LMPPs, CLPs and myeloid progenitors). The peripheral blood showed a decrease in mature cells and increase in the immature cells over time. Together, these findings suggest that the combination of ETV6-RUNX1 and IGF2BP1 promotes skewing of BM development towards an immature phenotype. These progenitor cells were hyperproliferative as seen by Ki67 staining. There was significant clonal expansion of these double positive cells. ETV6-RUNX1 overexpression alone led to a small but significant increase in progenitor output as reported previously [8, 9, 34]. This progenitor expansion appeared to be fuelled by both cell extrinsic and intrinsic mechanisms indicating that the combination led to an altered marrow niche favouring proliferation. IGF2BP1 has been shown to maintain stem cell properties by regulating HOXB4, ALDH1A1 and MYB in leukemic cell lines [13]. Histopathological analysis confirmed the hypercellular and disrupted architecture in the marrow and spleen and concomitant extramedullary hematopoiesis. ETV6-RUNX1 overexpression managed to increase endogenous Igf2bp1 levels in the bone marrow thus providing proof of our in-vitro finding.

Overexpression of ETV6-RUNX1 has been shown to increase HSCs with increased quiescence. Mature B-cell output has been known to be decreased with a developmental block at the pro-B cell stage [8]. In our combination experiments, Hardy fraction analysis showed an expansion of the small and large pre-B cell fractions indicating a developmental arrest. It is interesting to speculate a co-operative role for ETV6-RUNX1 and IGF2BP1 where the ETV6-RUNX1 promotes the progenitor expansion and prevents B-cell maturation which are then driven towards further expansion and leukemogenesis by the pro-oncogenic pathway genes stabilized by IGF2BP1.

Although the mice did not develop overt leukemia by 16 weeks, the observed features appear to mimic a pre-leukemic phenotype. Our findings appear to agree with previous studies which show that secondary hits are necessary for leukemogenesis even after ETV6-RUNX1 overexpression in mouse HSCs [9]. Due to its weak oncogenic nature, only a fraction of children with the ETV6-RUNX1 translocation go on to develop B-ALL. The positive feedback shown between ETV6-RUNX1 and IGF2BP1 may develop over time, leading to an incrementally altered transcriptome and the eventual emergence of a dominant transformed clone during leukemogenesis. By enforcing IGF2BP1 expression, we attempted to accelerate this process; and our data bears out this additive/synergistic relationship.

Our work uncovers a posttranscriptional, pro-oncogenic program driven by IGF2BP1 in ETV6-RUNX1 positive B-ALL. At the functional level, we have characterized and validated the differential activity of the non-canonical NFκB and PI3K-Akt pathways. These pathways are also reinforced by the feedback between ETV6-RUNX1 and IGF2BP1. With many of these pathways being druggable and a small molecule inhibitor for IGF2BP1 available, our work lays the foundation for novel combinatorial therapeutic approaches in ETV6-RUNX1 positive leukemias.

Conclusion

A murine model of ETV6-RUNX1 and IGF2BP1 overexpression has not been created before. This is the first time that such a model has been created using sequential retroviral infection. We have observed a novel pre-leukemic phenotype which has not been reported in any of the previous studies. Thus, our in-vivo mouse model supports the critical role of IGF2BP1 overexpression as a secondary hit and also confirmed the in-vitro finding of synergism between these two proteins. The next interesting thing will be to use IGF2BP1 as a therapeutic target for this particular subtype of leukemia.

Abbreviations

B-ALL

B-Acute Lymphoblastic Leukemia

IGF2BP1

Insulin like growth factor 2 binding protein 1

EFS

Event free survival

OS

Overall survival

qRT-PCR

quantitative real-time polymerase chain reaction

HSC

Hematopoietic stem cell

PGR

Prednisolone good responder

PPR

Prednisolone poor responder

RIP

RNA immunoprecipitation

MACS

Magnetic activated cell sorting

Declarations

Ethics approval and consent to participate

The study was approved by the Institutional Ethics and Biosafety Committees (IEC-1950/1.04.2016, RP-20/2016). Samples were collected after informed consent from a guardian and assent was taken from children >7 years of age in accordance with the Declaration of Helsinki regulations. In addition, some archival BM samples, preserved from previous studies, were also utilized after proper ethics clearance.

All mouse experimental procedures were conducted with the approval of the UCLA Chancellor’s Animal Research Committee (ARC).

Consent for publication

Not applicable.

Availability of Supporting Data:

All data generated or analyzed during this study are included in this published

article and its supplementary information files. The analyzed RNA-Seq and RIP-Seq data can be found in a data supplement. Raw reads are available in the SRA database with BioProject ID PRJNA837729.

Competing interests

All authors declare no potential conflict of interest.

Funding

This work was supported by the Wellcome Trust/DBT India Alliance Early Career Fellowship to JKP (IA/CPHE/15/1/502050). GS was supported by a DBT Senior Research Fellowship as well as a SERB Overseas Visiting Doctoral Research Fellowship. JS was supported by a DBT Senior Research Fellowship and AJ by a CSIR Senior Research Fellowship. DSR was supported by grants from the National Institutes of Health/ National Cancer Institute (R21CA197441, R01CA264986). TMT was supported by the Tumor Cell Biology Training Grant NIH T32 CA009056.

Authors’ Contributions

Conceptualization: JKP

Experimental Methodology and Data analysis: JKP, GS, TMT, JB, AKJ, CT, PC

Patient sample collection and clinical data analysis: AJ, GS, JS, SB, AS and AC

Bioinformatic Analysis: YT and DC carried out bioinformatic analysis.

Histopathology and data analysis: DSR

Project administration: JKP

Writing – original draft: GS, JKP

Writing – review and editing: All authors reviewed and edited the manuscript. They also provided critical feedback and helped in shaping the research and manuscript.

Acknowledgments

We thank Prof Kenneth Dorshkind and all our collaborators for vibrant discussions regarding the experiments and data. We would like to thank Dr. Anthony Ford, ICR London for the kind gift (pcDNA3.1-TEL-AML1 plasmid). The pHAGE6 vector was a kind gift from Dr Alejandro B. Balazs, Ragon Institute, MIT, MGH and Harvard. The pHAGE2-NFKB-TA-Luc-UBC-dTomato was a gift from Dr Darrell Kotton (Addgene plasmid # 49335) and pLKO5.sgRNA.EFS.tRFP was a gift from Dr Benjamin Ebert (Addgene plasmid # 57823).

Authors' information

1Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India

2Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, California, USA

3F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA

4Department of Laboratory Oncology, Dr B.R Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India

5 Department of Medical Oncology, Dr B.R Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India

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