Supplementary Methods, Figures 1 - 14, Tables 1 - 5 from Targeting Chromatin Regulators Inhibits Leukemogenic Gene Expression in <i>NPM1</i> Mutant Leukemia
Authors: Kühn Michael W.M. , Song Evelyn , Feng Zhaohui , Sinha Amit , Chen Chun-Wei , Deshpande Aniruddha J. , Cusan Monica , Farnoud Noushin , Mupo Annalisa , Grove Carolyn , Koche Richard , Bradner James E. , de Stanchina Elisa , Vassiliou George S. , Hoshii Takayuki , Armstrong Scott A. 
Published: April 2023

<p>Supplementary Figure 1. SURVEYOR assay assessing indel mutations of sgRNAs that target the N-HINGE-LOOP and the MBM-LBM locus. Supplementary Figure 2. Summary of negative selection experiments with sgRNAs targeting exons encoding specific MLL1 and MLL2 protein domains in a second independent OCI-AML3-Cas9 clone (OCI-AML3-Cas9-C8). Supplementary Figure 3. Dose responsive curves from cell viability assays of murine Npm1^CA/+ FIt3^ITD/+ after 7 days of MI-2-2 or MI-503 treatment and compared to MII-Af9 leukemia cells treated for 7 days with MI-503. Supplementary Figure 4. Apoptosis in OCI-AML3 cells treated for 7 days with MI-503 or DMSO as assessed by Annexin V / SYTOX blue staining. Supplementary Figure 5. Cytospins from cultured murine Npm1^CA/+ FIt3^ITD/+ comparing cells treated for 7 days with MI-503 versus DMSO. Supplementary Figure 6. Kaplan-Meier survival estimates from mice transplanted with secondary Npm1^CA/+ FIt3^ITD/+ leukemia cells (n=5 mice/group) and treated in vivo for ten consecutive days with either MI-503 or vehicle control. Supplementary Figure 7. Kaplan-Meier estimates of mice transplanted with secondary Npm1^CA/+ FIt3^ITD/+ leukemia cells (n=10 mice/group) that had been pretreated with either 10 days of EPS4777 [10 microM] or vehicle prior to transplantation. Supplementary Figure 8. Schematic of RNA sequencing detecting an MLL-AF6 fusion transcript in the DNMT3A mutated AML cell line OCI-AML2. Supplementary Figure 9. RNA sequencing of the OCI-AML2 cells reveals a HOX gene expression pattern as it is typically found in MLL-rearranged leukemias with predominant HOXA and MEIS1 expression but lack of HOXB cluster expression. Supplementary Figure 10. Isobolograms showing drug synergism for (A) MI-2-2 and EPZ4777 in the OCI-AML3 cells and (B) MI-503 and EPZ4777 in the murine Npm1^CA/+ FIt3^ITD/+ cells. Supplementary Figure 11. ChIP-PCR assessing RNA-Polymerase II enrichment after vehicle, single drug (MI-503 or EPZ4777) or combination treatment at the MEIS1 locus. Supplementary Figure 12. Cytologic analysis of human OCI-AML3 cells treated with DMSO, MI-2-2 (12 microM), EPZ4777 (10 microM), or combination therapy (MI-2-2 + EPZ4777). Supplementary Figure 13. Number of morphologically differentiated cells of two primary AML samples (ID1 and ID3) that had been treated in a human co-culture assay for 10 days with DMSO, EPZ4777, MI-503, or EPZ4777 and MI-503 (combo). Supplementary Figure 14. Engraftment of Npm1^CA/+ Rosa^SB/+ leukemia cells in the peripheral blood of tertiary recipient animals 22 days after transplantations. Supplementary Table 1. MLL1 single guide RNAs (sgRNA). Supplementary Table 2. MLL2 single guide RNAs. Supplementary Table 3. Synergy analysis of OCI-AML3 cells (day 7). Supplementary Table 4. Synergy analysis of Npm1^CA/+ FIt3^ITD/+ leukemias (day 11). Supplementary Table 5. Primary AML samples.</p>