Additional file 1 of Immunoproteasome function maintains oncogenic gene expression in KMT2A-complex driven leukemia
Creators: name: Tubío-Santamaría, Nuria
name: Jayavelu, Ashok Kumar
name: Schnoeder, Tina M.
name: Eifert, Theresa
name: Hsu, Chen-Jen
name: Perner, Florian
name: Zhang, Qirui
name: Wenge, Daniela V.
name: Hansen, Fynn M.
name: Kirkpatrick, Joanna M.
name: Jyotsana, Nidhi
name: Lane, Steven W.
name: von Eyss, Björn
name: Deshpande, Aniruddha J.
name: Kühn, Michael W. M.
name: Schwaller, Juerg
name: Cammann, Clemens
name: Seifert, Ulrike
name: Ebstein, Frédéric
name: Krüger, Elke
name: Hochhaus, Andreas
name: Heuser, Michael
name: Ori, Alessandro
name: Mann, Matthias
name: Armstrong, Scott A.
name: Heidel, Florian H.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology

description: Additional file 1: Fig. S1. (A) Western Blotting showing expression of PSMB8, PSMB9 and PSMB10 in KMT2A-r (ML-2, THP-1, MV-4;11, KOPN-8, MOLM-13) and non-KMT2A-r (HL-60, K-562) cell lines. Hela cells were used as a negative control, since they do not express immunoproteasome, and T cells as a positive control. (B) Violin plots displaying log2-fold protein expression as assessed by proteome analysis (Jayavelu et al., Cancer Cell, 2022; PXD022894) of PSMB8, PSMB9 and PSMB10 in different KMT2A-r and non-KMT2A-r cell lines. Mann-Whitney U test. (C) CRISPR-Cas9 cell competition assay showing the effect of deletion of each catalytic immunoproteasome subunit over time. Catalytic immunoproteasome subunits were knocked out using different single guide RNAs (5 specific for PSMB8; 3 specific for PSMB9 and PSMB10, respectively) in human KMT2A-r AML cells (MOLM-13) and the chimerism of knockout and wildtype cells over time is visualized in the graphs. Each blue line represents the chimerism at day 0, 3, 6, 9, and 12. A decrease in the % of RFP+ cells (shown on the Y axis) over time, as seen most pronounced for PSMB8, reflects a competitive disadvantage of cells that harbor the respective knockout. Single guide RNAs (red lines) against the essential gene RPA3 were used as a positive control and non-targeting guide against Luciferase as a negative control (black line). (D) Percentage of Annexin+ cells (containing Annexin+-SYTOX®Blue+ and Annexin+-SYTOX®Blue- populations measured by flow cytometry) in KMT2Ar cell lines (MOLM-13, THP-1, MONO-MAC-6, KOPN-8, ML-2) transduced with shRNAs targeting PSMB8 or a non-targeting control (shNT) at day 6 post-infection. n=3-5 independent experiments; mean with SD; paired Student t test. (E) Growth curves depicting cell counting after trypan blue exclusion of MOLM-13 cells and MOLM-13 PSMB8 overexpressing cells (+PSMB8) transduced with shRNAs targeting PSMB8 or a non-targeting control (shNT). n=3 independent experiments; mean with SD. (F) Representative pictures of colonies from MOLM-13 and ML-2 cells transduced with PSMB8 shRNAs or shNT. Scale bars, 200 µm. Representative Western Blotting plots of MOLM-13 and ML-2 cells confirming PSMB8 deletion at day 4 post-infection before transplantation into NSGS recipient mice. (G) Representative Western Blotting plots of MOLM-13 and ML-2 cells confirming PSMB8 deletion at day 4 post-infection before transplantation into NSGS recipient mice. Fig. S2. (A) Violin plots showing the percentage of cells detected in S phase in MOLM-13, ML-2, KOPN-8 and MV-4;11, cells after treatment with 100nM PR-957 or DMSO for 24 hours. Samples were labeled for flow cytometry-based analysis of cell cycle using the Click-iT® EdU assay. n=3 independent experiments; paired Student t test. (B) Representative flow cytometry plots from Click-iT® EdU assay. (C) Schematic representation of patient derived xenografts (PDX). (D) Violin plots depicting % of hCD45+ cells in peripheral blood in PDX models of KMT2A::MLLT3 and KMT2A::AFF1. 2-way ANOVA. (E) Violin plots of hCD45+ cells in spleen and bone marrow at the time of sacrifice. Mann-Whitney U test. Fig. S3. (A) Serial re-plating to assess colony formation in methylcellulose using murine LSK (Lin- Sca1+ c-Kit+) cells isolated from LMP7+/+ or LMP7-/- mice and transformed with KMT2A::MLLT3 or KMT2A::MLLT1. n=4 independent experiments; mean with SD; paired Student t test. (B) Schematic representation showing the transplantation of KMT2A::MLLT3 retrovirally transformed LSKs from LMP7+/+ or LMP7-/- mice. (C) Schematic representation describing the transplantation of i-KMT2A::MLLT1 BM cells lentivirally transduced with shRNA1 or shRNA4 against LMP7 or a non-targeting control (shNT) into CD45.1 recipient mice. Diet of the donor mice was supplemented with Doxycycline (DOX; 0,545 g/kg) for 2 weeks to induce KMT2A::MLLT1 expression. Recipient mice were also kept with DOX supplemented food. (D) Violin plots depicting percentage of CD45.2+ cells in peripheral blood of CD45.1 recipient mice from sh1 LMP7 (n=4), sh4 LMP7 (n=4) or shNT (n=4) transformed i-KMT2A::MLLT1. One cohort; 2-way ANOVA. (E) Kaplan-Meier survival curves of CD45.1 recipient mice transplanted with 1x106 cells as shown in S3C. One cohort; Mantel-Cox test. (F) Spleen Colony Formation Assay in vivo (CFU-S12): Spleen colony numbers 12 days after injection of 100 LSK cells isolated from LMP7+/+ (n=10) or LMP7-/- (n=10) mice. Two independent cohorts; Mann-Whitney U test (G) Schematic representation depicting competitive repopulation assay to investigate the effects of LMP7 depletion on normal hematopoietic stem- and progenitor cells (HSPCs). Fig. S4. (A) Representative pictures of colonies from murine LSK cells transformed with KMT2A::MLLT3/KRAS, KMT2A::MLLT4 or AML1-ETO/KRAS at week 2. Scale bars, 200 µm. (B) Schematic representation of in vivo PR-957 vs NaCl 0.9% treatment in C57BL/6 mice transplanted with KMT2A::MLLT3 leukemic cells. Subsequent secondary transplantation of whole bone marrow cells into secondary recipients. (C) Pictures of tissue sections from liver, lung and spleen of in vivo PR-957 vs NaCl treated mice at the time of sacrifice. (D) Representative flow cytometry plots with the gating strategy for the analysis of granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), common myeloid progenitors (CMPs), hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) in the competitive repopulation assay. FSA: Forward Scatter Area; SSA: Side Scatter Area; FSH: Forward Scatter Height; FSW: Forward Scatter Width; SSH: Side Scatter Height; SSW: Side Scatter Width; LK: Lineage- cKit+; LSK: Lineage- Sca1+ cKit+. Fig. S5. (A) Volcano plot of differentially regulated genes in RNA-sequencing. 100nM PR-957 vs. DMSO, 72h, MOLM-13. Upregulated (red; FC&gt;1.5, p&lt;0.05) and downregulated (blue; FC&lt;-1.5, p&lt;0.05). (B) Heatmaps displaying H3K27ac, H3K27me3, H3K4me1 and H3K4me3 Cut&amp;Tag signal mapping to a 2-kb window around TSS. 100nM PR-957 vs. DMSO, 48h, MOLM-13. (C) Integrative Genomics Viewer (IGV) tracks from Cut&amp;Tag-sequencing data in MOLM-13 cells depicting binding of H3K27ac, H3K27me3, H3K4me1 and H3K4me3 after DMSO or PR-957 treatment at HOXA and MEIS1 loci. (D) Volcano plot of differentially accessible regions in ATAC-seq. 100nM PR-957 vs. DMSO, 72h, MOLM-13. Upregulated (red; FC&gt;2, p&lt;0.05) and downregulated (blue; FC&lt;-2, p&lt;0.05). (E) Stacked bar plot depicting genomic distribution of PSMB8 ATAC-seq peaks. 100nM PR-957 vs. DMSO, 72h, MOLM-13. (F) Schematic representation of the genome-wide CRISPR-Cas9 screen. (G) Volcano plots showing the distribution of genes being enriched (positive beta-scores) or depleted (negative beta-scores) in the genome-wide CRISPR-Cas9 screen in PR-957 treated MOLM-13 cells and DMSO diluent control. (H-I) Western Blotting showing expression of BASP1 in nuclear (N) and cytoplasmic (C) fractions of MOLM-13, MV-4;11, KOPN-8 and ML-2 cells. (H) BTZ (4nM) vs. DMSO, 72h. (I) CFZ (4nM) vs. DMSO, 72h. (J) Integrative Genomics Viewer (IGV) tracks from Cut&amp;Run-sequencing data in MOLM-13 cells depicting binding of BASP1 after DMSO or PR-957 treatment and of H3K4me3 after DMSO treatment at the KMT2A, MYC, JUN and GNAS loci. Fig. S6. (A) Western Blotting confirming BASP1 overexpression after transduction with a pLEX vector containing the sequence of human BASP1 with an HA tag (BASP1) or an empty vector with the HA tag (EV) as a control in MOLM-13, MV-4;11, ML-2 and KOPN-8 cells. Samples were collected at day 6 post-infection. (B) Percentage of Annexin+ cells of KMT2A-r cell lines overexpressing BASP1 or control cells at day 6 post-infection. n=4 independent experiments; mean with SD; paired Student t test. (C) Growth curves of MOLM-13-deadCas9 (MOLM-13-dCas9) and MV-4;11-dCas9 cells containing the MS2-P65-HSF1 activator helper complex transduced with sgRNAs designed to target the promoter region of BASP1 (sgRNA3a BASP1 and sgRNA5a BASP1) or sgRNA Luciferase as a negative control. n=4 independent experiments; mean with SD; 2-way ANOVA. (D) Relative BASP1 mRNA expression in BASP1-overexpressing cells (MOLM-13-dCas9 and MV-4;11-dCas9) compared to sgRNA Luciferase cells assessed by Real Time Quantitative PCR (RT-qPCR). n=4 independent experiments; mean with SD. (E) Western Blotting confirming BASP1 overexpression after transduction with pLEX-BASP1 (BASP1) or pLEX-EV (EV) in MOLM-13 and MV-4;11 cells. Samples were collected 96 hours after transduction and before transplantation into recipient mice. Fig. S7. (A) Western Blotting quantification of c-MYC, MEF2C, FLT3 (mature), FLT3 (premature), PBX3, PSMB5 and PSMB8 protein expression in MOLM-13 cells treated with 100nM PR-957, 1μM MI-503, a combination of both or DMSO. n=3-4 independent experiments; mean with SD; paired Student t test, (B) Percentage of Annexin+ cells in MOLM-13, MV-4;11, ML-2, KOPN-8 and OCI-AML3 cells after treatment with 20nM PR-957, 200nM MI-503, a combination of both or DMSO as diluent control at day 8 post-infection. n=5 independent experiments; mean with SD; paired Student t test. (C) Kaplan-Meier survival curves of NXG recipient mice transplanted with limiting numbers (2x106, 2x105, 2x104) of whole bone marrow cells from in vivo treated mice (DMSO, MI-503, MI-503 + PR-957). Two independent cohorts; Mantel-Cox test. (D) Violin plots showing percentage of hCD45+ cells in bone marrow (BM) at the time of sacrifice. Fig. S8. (A) Negative selection cell competition assay using MV-4;11 Menin-wildtype (BFP+) cells and resistant MV4;11 Menin M327I or T349M (RFP+) cells (as published in Perner et al. Nature 2023). Chimerism of RFP+ mutant clones over BFP+ Menin-wildtype cells is visualized for a total of 9 days upon exposure to PR-957(100nM), Revumenib (50nM), MI503 (1μM) or DMSO as a control. Table S1. Flow cytometry and western blot antibodies used in this study. Table S2. shRNA sequences used in this study. Table S3. sgRNAs for CRISPRa used in this study. Table S4. Primer sequences for RT-qPCR used in this study.