Supplementary Data from MOZ and Menin–MLL Complexes Are Complementary Regulators of Chromatin Association and Transcriptional Output in Gastrointestinal Stromal Tumor

Missense mutants of p53, such as the frequent hotspot variant R248Q, exert a dominant-negative effect (DNE) on wild-type (WT) p53 in cancer cells with monoallelic TP53 mutations. However, the precise functional and molecular mechanisms of the DNE have remained elusive due to a lack of appropriate model systems. In this study, we developed a variety of model systems, including CRISPR-edited human isogenic cell lines and transcriptional reporter cell lines, and targeted protein degradation assays that were combined with functional and molecular analyses to functionally characterize the DNE. Formation of heterotetramers between R248Q and WT p53 impaired proper WT p53 functionality by preventing DNA binding and subsequent target gene transactivation. Furthermore, the markedly increased protein half-life of R248Q led to supraphysiologic levels of R248Q, which was critically required for the DNE. Drug-induced targeted protein degradation of R248Q to lower the R248Q:WT ratio restored the transcriptional activity of WT p53, induced antiproliferative effects in cancer cells in vitro, and elicited strong therapeutic activity in vivo. Together, this study provides mechanistic insights into the DNE of p53 missense mutants and indicates that the DNE represents a promising therapeutic target.Significance: Heterotetramerization between R248Q mutant and wild-type p53 in conjunction with supraphysiologic p53R248Q accumulation underlies the dominant-negative effect, highlighting the need to develop pharmacologic strategies to decrease the elevated R248Q:WT ratio.See related commentary by Gencel-Augusto and Lozano, p. 1955

Supplementary Figures S1-S25

Processed data for S222A+ and S222A- cells by RNA and ATAC sequencing

Processed data for MRT199665 and DMSO treated OCI-AML2 cells by RNA sequencing

Supplementary Data from MLL-Rearranged B Lymphoblastic Leukemias Selectively Express the Immunoregulatory Carbohydrate-Binding Protein Galectin-1

Supplementary Tables S1-S3 and Supplementary Figures S1-S6. Table S1 - EC50 values for B-cell malignancies treated with HDAC inhibitors of varying isoform selectivity Table S2 - EC50 values for B-ALL cell line treated with a panel of HDAC inhibitors Table S3 - statistics for cell growth assay in Figure 3 Figure S1 - In vitro survival and PD for B-ALL cells treated with LAQ824 Figure S2 - Western analyses of B-ALL cells treated with MS275 Figure S3 - Western blots, cell growth and cell cycle analysis for B-ALL cells transduced with HDAC1,2, or 3 specific shRNA Figure S4 - Western analyses, cell growth and survival of B-ALL cell treated with Merck60 Figure S5 - Survival and western blots for B-ALL cells treated with HDACi with an apoptosis inhibitor. Figure S6 - Western blots to show DNA damage caused by HDACi or doxorubicin in Merck60 insensitive multiple myeloma cell lines.

Supplementary Table S1-S3 Legends and Supplementary Figure S1-S6 Legends. Legends for supplementary tables S1-S3 and supplementary figures S1-S6 which support data in the manuscript, but could not be included due to space constraints.

EDITORIAL article Front. Physiol., 14 March 2023Sec. Integrative Physiology Volume 14 - 2023 | https://doi.org/10.3389/fphys.2023.1179083

Abstract Menin interacts with oncogenic MLL1-fusion proteins, and small molecules that disrupt these associations are in clinical trials for leukemia treatment. By integrating chromatin-focused and genome-wide CRISPR screens with genetic, pharmacologic, and biochemical approaches, we discovered a conserved molecular switch between the MLL1–Menin and MLL3/4–UTX chromatin-modifying complexes that dictates response to Menin–MLL inhibitors. MLL1–Menin safeguards leukemia survival by impeding the binding of the MLL3/4–UTX complex at a subset of target gene promoters. Disrupting the Menin–MLL1 interaction triggers UTX-dependent transcriptional activation of a tumor-suppressive program that dictates therapeutic responses in murine and human leukemia. Therapeutic reactivation of this program using CDK4/6 inhibitors mitigates treatment resistance in leukemia cells that are insensitive to Menin inhibitors. These findings shed light on novel functions of evolutionarily conserved epigenetic mediators like MLL1–Menin and MLL3/4–UTX and are relevant to understand and target molecular pathways determining therapeutic responses in ongoing clinical trials. Significance: Menin–MLL inhibitors silence a canonical HOX- and MEIS1-dependent oncogenic gene expression program in leukemia. We discovered a parallel, noncanonical transcriptional program involving tumor suppressor genes that are repressed in Menin–MLL inhibitor–resistant leukemia cells but that can be reactivated upon combinatorial treatment with CDK4/6 inhibitors to augment therapy responses. This article is highlighted in the In This Issue feature, p. 1

Table of phosphopeptides identified in a screen for primary induction failure compared to complete remission AML

Extended methods, supplementary figures and supplementary tables.

Table of results for recombinant protein kinase screen for pS222 MEF2C

Macrophages, the key cells of innate immunity, possess wide phenotypical and functional heterogeneity. In vitro studies showed that microenvironment signals could induce the so-called polarization of macrophages into two phenotypes: classically activated macrophages (M1) or alternatively activated macrophages (M2). Functionally, they are considered as proinflammatory and anti-inflammatory/pro-regenerative, respectively. However, in vivo studies into macrophage states revealed a continuum of phenotypes from M1 to M2 state instead of the clearly distinguished extreme phenotypes. An important role in determining the type of polarization of macrophages is played by energy metabolism, including the activity of oxidative phosphorylation. In this regard, hypoxia and ischemia that affect cellular energetics can modulate macrophage polarization. Here, we overview the data on macrophage polarization during metabolic shift-associated pathologies including ischemia and ischemia/reperfusion in various organs and discuss the role of energy metabolism potentially triggering the macrophage polarization.

Abstract Gastrointestinal stromal tumor (GIST) is commonly characterized by activating mutations in the receptor tyrosine kinase KIT. Tyrosine kinase inhibitors are the only approved therapy for GIST, and complementary treatment strategies are urgently needed. As GIST lacks oncogene amplification and relies upon an established network of transcription factors, we hypothesized that unique chromatin-modifying enzymes are essential in orchestrating the GIST epigenome. We identified through genome-scale CRISPR screening that MOZ and Menin–MLL chromatin regulatory complexes are cooperative and unique dependencies in GIST. These complexes were enriched at GIST-relevant genes and regulated their transcription. Inhibition of MOZ and Menin–MLL complexes decreased GIST cell proliferation by disrupting interactions with transcriptional/chromatin regulators, such as DOT1L. MOZ and Menin inhibition caused significant reductions in tumor burden in vivo, with superior effects observed with combined Menin and KIT inhibition. These results define unique chromatin regulatory dependencies in GIST and identify potential therapeutic strategies for clinical application. Significance: Although many malignancies rely on oncogene amplification, GIST instead depends upon epigenetic regulation of KIT and other essential genes. Utilizing genome-scale CRISPR dependency screens, we identified complementary chromatin-modifying complexes essential to GIST and characterize the consequences of their disruption, elucidating a novel therapeutic approach to this disease. This article is highlighted in the In This Issue feature, p. 1599

The initiating mutations that contribute to cancer development are sometimes present in premalignant cells. Whether therapies targeting these mutations can eradicate premalignant cells is unclear. Acute myeloid leukemia (AML) is an attractive system for investigating the effect of preventative treatment because this disease is often preceded by a premalignant state (clonal hematopoiesis or myelodysplastic syndrome). In Npm1c/Dnmt3a mutant knock-in mice, a model of AML development, leukemia is preceded by a period of extended myeloid progenitor cell proliferation and self-renewal. We found that this self-renewal can be reversed by oral administration of a small molecule (VTP-50469) that targets the MLL1-Menin chromatin complex. These preclinical results support the hypothesis that individuals at high risk of developing AML might benefit from targeted epigenetic therapy in a preventative setting.