Fig 2: NF1 is a target gene of miR-103a-3. (A) Sequence alignment of miR-103a-3p with the 3′-UTR of NF1 gene. The red labeled nucleotides represent point mutations in the 3′-UTR sequence. miR-103a-3p negatively regulates NF1 expression at both the (B) protein and (C) mRNA levels. A549 cells were transfected with miR-103a-3p mimic or inhibitor. After 72 h of transfection, cells were subjected to reverse transcription-quantitative PCR and western blot analysis. (D) 3′-UTR luciferase reporter assay for NF1. Each miR-103a-3p binding site of NF1 3′-UTR was cloned into luciferase reporter constructs and transfected into A549 cells with miR-103a-3p mimic or control oligonucleotides. The luciferase assay results revealed that the signal was significantly decreased following transfection with the third site of the NF1 3′-UTR. (E) The third NF1 3′-UTR binding site inhibits the expression of firefly luciferin. The indicated luciferin plasmid was transfected with miR-103a-3p mimic or control oligonucleotides. *P<0.05 and **P<0.01 with comparisons indicated by lines. NF1, neurofibromin 1; miR, microRNA; 3′-UTR, 3′-untranslated region; NC, negative control; mut, mutant.

Fig 3: Knockdown of NF1 and CUL3 drive vemurafenib resistance in A375 cells. (A,D) The efficiency of CUL3 (A) and NF1 (D) knockdown in A375 cells was confirmed by RT-PCR and western blot [*p < 0.05; **p < 0.01; ****p < 0.0001 (One-way ANOVA followed by Bonferroni's multiple comparisons test)]. (B,E) Sensitivity to vemurafenib was evaluated in short-term (72 h) dose response assay in CUL3 (B) and NF1 (E) A375 knockdown cells. (C,F) Sensitivity to vemurafenib was evaluated in long-term (70 days) growth assay in CUL3 (C) and NF1 (F) A375 knockdown cells [****p < 0.0001 (Two-way ANOVA followed by Bonferroni's multiple comparisons test)]. n ≥ 3 for each experiment. See Supplementary Figure 6 for original blots.

Fig 4: Identification of pyrvinium pamoate as anti-leukemic compound active in RAS+ AML.A Schematic representation of high-density pharmacological screen in NF1 KO TF-1 cells. B First screen using the PCL library of 1280 compounds at 10μM and CellTiter-Glo® cell viability reagent after 72 h of incubation. Results are represented for each compound (identified by a single dot) by the relation between their robust Z-score value (RZ-score) in Y-axis and the percentage of cell growth in X-axis. Compounds with a RZ-score ≤ −5 (retained for further analysis) are highlighted in red. C Second screen performed with serial dilutions of the top-60 compounds from the first screen in NF1 KO TF-1 cells. Results are presented for each compound illustrated by a dot as the correspondence between their median effective dose (ED50, represented using a Log10 scale) and drug sensitivity score (DSS). The best hits are highlighted in red, and the classical AML chemotherapies (daunorubicin and cytarabine) are highlighted in orange. D Dose-range experiments using log-dilutions (10−5 to 10−8 M) of pyrvinium pamoate in CTR (non-targeting sgRNA), NF1KO-1, NF1KO_2 and NRASG12D Ba/F3 cells (left panel) or TF-1 (right panel) cells during 48 h. * indicate culture in the presence of IL-3 (Ba/F3 cells) or GM-CSF (TF-1 cells). Results in the control condition of each compound were normalized to all control conditions across each plate.

Fig 5: (A). The top row details the amino acid sequence of the fragment of human NF1 protein that affinity purified from WT HEK293 cells with the tagged mNf1 cDNA. The second row depicts the homologous mouse neurofibromin protein sequence and highlights the non-conserved amino acid residue T2489A. (B). Reciprocal immunoprecipitation of the tagged murine and human cDNAs. The left-hand side shows blots of input lysates after cell transfections with each indicated cDNA prior to affinity purification. The right-hand side shows blots after affinity purification. The left two columns show the Strep tag affinity purification and the right two columns show the Halo tag affinity purification. WT indicates the co-transfection of both cDNAs into HEK293 NF1 null cells; while EV indicates the empty vector transfection into the same cells. The first row of immunoblots is the visualization of neurofibromin. The second row shows the His tag from the mNf1 cDNA. The third row depicts the Halo tag from the hNF1 cDNA. The His tag is visible in the Halo immunoprecipitation WT lane and the Halo tag is visible in the Strep tag affinity purification WT lane, indicating that the two tagged neurofibromins are purifying the reciprocal neurofibromin when the individual affinity purification is completed.