Additional file 1 of RNA binding protein IGF2BP1 synergizes with ETV6-RUNX1 to drive oncogenic signaling in B-cell Acute Lymphoblastic Leukemia
Creators: name: Sharma, Gunjan
name: Tran, Tiffany M.
name: Bansal, Ishu
name: Beg, Mohammad Sabique
name: Bhardwaj, Ruchi
name: Bassi, Jaspal
name: Tan, Yuande
name: Jaiswal, Amit Kumar
name: Tso, Christine
name: Jain, Ayushi
name: Singh, Jay
name: Chattopadhyay, Parthaprasad
name: Singh, Archna
name: Chopra, Anita
name: Bakhshi, Sameer
name: Casero, David
name: Rao, Dinesh S.
name: Palanichamy, Jayanth Kumar
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Plant Biology
subject: Computational Biology

description: Additional file 1: Figure S1. Relative Expression of ETV6-RUNX1 in multiple ETV6:: RUNX1 positive and negative cell lines. POLR2A and HGPRT were used as internal controls. Figure S2. SETEN analysis of RIP-Seq targets of IGF2BP1 using the A) MALA Cards B) GO Biological Processes and C) GO Molecular Function databases D) UCSC Genome browser snapshots of CDK6, MDM2, IL6ST, NFAT5 showing the enrichment of these targets in the IGF2BP1 precipitated fraction. Figure S3. GSEA (Hallmark pathways) of differentially expressed genes between ETV6::RUNX1 positive (n=14) and TCF3::PBX1 positive (n=15) subtypes of B-ALL patient samples showing activated and suppressed pathways with enrichment scores. Figure S4. GSEA (Hallmark pathways) of differentially expressed genes between ETV6::RUNX1 positive (n=14) and KMT2A translocated (n=15) subtypes of B-ALL patient samples showing activated and suppressed pathways with enrichment scores. Figure S5. A comparison of selected genes’ expression in the TNFα via NFκB and PI3K-AKT pathways from the a) IGF2BP1 knockout dataset (log2Fc; &lt;1 refers to downregulation) b and c) analysis of public microarray based dataset of ETV6::RUNX1 vs TCF3::PBX1 patient samples and ETV6::RUNX1 vs KMT2A rearranged patient samples (log2Fc) d) RIP enrichment based on fold change of IGF2BP1 immunoprecipitated RNA to input RNA levels and e) public dataset of differentially expressed genes between ETV6::RUNX1 positive B-ALL patients (n=4) and CD19 positive B-cells (n=2) (log2Fc). The TNFα via NFκB pathway targets are downregulated after IGF2BP1 KO, enriched in the RIP data and the ETV6::RUNX1 positive tumors showing that these are direct targets bound and stabilized by IGF2BP1. The PI3K pathway targets are bound by IGF2BP1 and enriched in the tumors but not downregulated after IGF2BP1 KO implying a multifactorial regulation of their mRNA stability. Figure S6. A) Validation of RIP targets of IGF2BP1 using the public TARGET dataset (E6R1+ n=20, Other translocations n=27, Hyperdiploid n=55, No Known Sentinel Translocations n=95) B) Correlation between IGF2BP1 and its target genes in the patient samples with high IGF2BP1 expression (n=29). Figure S7. A)-B) Correlation between IGF2BP1 and its target genes in the TARGET cohort (n=29) C) Expression of BCL2 and MYB after IGF2BP1 KO and their RIP enrichment from our datasets. Figure S8. Cell viability in (A-D) ETV6-RUNX1 translocation positive cell lines along with E) RL (Non-Hodgkin lymphoma) and F) Jurkat, a T-ALL cell line after functional inhibition of IGF2BP1 using BTYNB treatment. Cell viability measured by MTS assay (AC) and trypan blue exclusion assay (D-F). Figure S9. Expression of IGF2BP1 and ETV6::RUNX1 in ETV6-RUNX1 translocation positive cell lines as determined by qRT-PCR after BTYNB treatment (5 µM). Figure S10. Expression of IGF2BP1 targets of NFKB and PI3K-AKT signalling pathway after IGF2BP1 inhibition by BTYNB (5/10µM) treatment compared to DMSO-treated cells. (AT1, AT2, UOCB6 – ETV6-RUNX1 translocation positive cell lines and RS4;11, NALM6, THP1 – ETV6-RUNX1 translocation negative cell lines). POLR2A and HGPRT were used as internal controls. Figure S11. Western Blots showing reduced protein expression of IGF2BP1 targets of NFkB (IKKα and NFκβ) and PI3K-AKT (AKT, MDM2, GSK3β and PDK1) signalling pathway in ETV6-RUNX1 positive cell lines after IGF2BP1 inhibition by BTYNB. Densitometric values are provided above each blot normalized to the DMSO control. GAPDH was used as internal control. (Note: For NFkB, densitometry was performed using p50, the functional sub-unit.). Figure S12. Figure S11: Western Blots showing no change in protein expression of IGF2BP1 targets of NFkB (IKKα and NFκβ) and PI3K-AKT (AKT, MDM2, GSK3β and PDK1) signalling pathway in ETV6-RUNX1 positive cell lines after IGF2BP1 inhibition by BTYNB. Densitometric values are provided above each blot normalized to the DMSO control. GAPDH was used as internal control. (Note: For NFkB, densitometry was performed using p50, the functional sub-unit; RS4;11 expressed extremely low levels of p-GSK3β (Ser9) to detect and analyze). Figure S13. A) Schematic of cloning the IGF2BP1 CDS in a pHAGE6 based lentiviral vector and cloning the ETV6::RUNX1 fusion junction in a dual luciferase pMirGlo vector B) Schematic showing regions targeted by ETV6::RUNX1 targeted guide RNAs which were then cloned in pLKO5-tRFP vector C) Schematic of ETV6::RUNX1 fusion transcript overexpressing retroviral vector. Figure S14. A) Schematic of the bone marrow transplant experiment B) Vector schematic of the ETV6::RUNX1 fusion transcript and IGF2BP1 overexpressing murine retroviral vectors C) Western blot showing overexpression of IGF2BP1 in 293T cells after MICHIGF2BP1 transfection; GAPDH was used as a loading control D) qRT-PCR to quantify overexpression of ETV6::RUNX1 in 293T cells after MIG-ETV6::RUNX1 transfection. Figure S15. A) Time course graph showing increasing clonal expansion in the peripheral blood of IGF2BP1+ETV6::RUNX1 combination group with time (t-test; p * &lt; 0.05, ** &lt; 0.01, *** &lt; 0.005, **** &lt; 0.0001) B) Representative FACS plots indicating clonal expansion. Figure S16. A-H) Quantification of various hematological parameters from the peripheral blood of mice at 16 weeks (t-test; p * &lt; 0.05, ** &lt; 0.01, *** &lt; 0.005, **** &lt;0.0001). Figure S17. Quantification of immature B-cell populations (Hardy Fractions) in the bone marrow (absolute counts and percentages) (t-test; p * &lt; 0.05, ** &lt; 0.01, *** &lt; 0.005, **** &lt;0.0001). Figure S18. Quantification of absolute counts of myeloid lineage committed progenitors (CMP, GMP and MEP) and their respective percentages in the bone marrow (t-test; p *&lt; 0.05, ** &lt; 0.01, *** &lt; 0.005, **** &lt;0.0001). Figure S19. Subset analysis of various progenitor populations in different fractions of the bone marrow: GFP+mCherry+ (double positive) and GFP-mCherry- (double negative) fractions; This revealed a cell intrinsic and extrinsic role in the IGF2BP1+ETV6::RUNX1 combination group favoring progenitor expansion (t-test; p *&lt;0.05, ** &lt; 0.01, *** &lt; 0.005, **** &lt; 0.0001). Figure S20. A) Spleen weights of mice belonging to different groups at week 16 B) Quantification of double positive ratio in the splenocytes and C) Lin- population by the ratio of GFP+ mCherry+ double positive cells D) Quantification of HSCs in the spleen (t-test; p *&lt; 0.05, ** &lt; 0.01, *** &lt; 0.005, **** &lt; 0.0001) E) Histological analysis of spleens of mice belonging to different groups; Controls show a prominent germinal center and marginal zone with clear white and red pulp distinction; Red pulp expansion, smaller germinal centers and loss of architecture is seen in IGF2BP1+ETV6::RUNX1 combination (200X). Supplementary Table 1. List of primers used in this study. Supplementary Table 2. List of guide RNAs used in this study. Supplementary Table 3. Surface markers for hematopoietic progenitors. Supplementary Table 4. Hardy fractions Surface markers. Supplementary Table 5. List of antibodies used in flowcytometry.