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Epigenetic programs are dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells. This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene-expression programs. We show that KAT6A is the initiator of a newly described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyl-lysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation. Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small-molecule inhibitors could be of high therapeutic interest for mono-therapy or combinatorial differentiation-based treatment of AML. Significance: AML is a poor-prognosis disease characterized by differentiation blockade. Through a cell-fate CRISPR screen, we identified KAT6A as a novel regulator of AML cell differentiation. Mechanistically, KAT6A cooperates with ENL in a “writer–reader” epigenetic transcriptional control module. These results uncover a new epigenetic dependency and therapeutic opportunity in AML. This article is highlighted in the In This Issue feature, p. 587

Supplementary Data from KAT6A and ENL Form an Epigenetic Transcriptional Control Module to Drive Critical Leukemogenic Gene-Expression Programs

Epigenetic programs are dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells. This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene-expression programs. We show that KAT6A is the initiator of a newly described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyl-lysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation. Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small-molecule inhibitors could be of high therapeutic interest for mono-therapy or combinatorial differentiation-based treatment of AML.Significance:AML is a poor-prognosis disease characterized by differentiation blockade. Through a cell-fate CRISPR screen, we identified KAT6A as a novel regulator of AML cell differentiation. Mechanistically, KAT6A cooperates with ENL in a “writer–reader” epigenetic transcriptional control module. These results uncover a new epigenetic dependency and therapeutic opportunity in AML.This article is highlighted in the In This Issue feature, p. 587

Protein quality control systems are crucial for cellular function and organismal health. At present, most known protein quality control systems are multicomponent machineries that operate via ATP-regulated interactions with non-native proteins to prevent aggregation and promote folding1, and few systems that can broadly enable protein folding by a different mechanism have been identified. Moreover, proteins that contain the extensively charged poly-Asp/Glu (polyD/E) region are common in eukaryotic proteomes2, but their biochemical activities remain undefined. Here we show that DAXX, a polyD/E protein that has been implicated in diverse cellular processes3–10, possesses several protein-folding activities. DAXX prevents aggregation, solubilizes pre-existing aggregates and unfolds misfolded species of model substrates and neurodegeneration-associated proteins. Notably, DAXX effectively prevents and reverses aggregation of its in vivo-validated client proteins, the tumour suppressor p53 and its principal antagonist MDM2. DAXX can also restore native conformation and function to tumour-associated, aggregation-prone p53 mutants, reducing their oncogenic properties. These DAXX activities are ATP-independent and instead rely on the polyD/E region. Other polyD/E proteins, including ANP32A and SET, can also function as stand-alone, ATP-independent molecular chaperones, disaggregases and unfoldases. Thus, polyD/E proteins probably constitute a multifunctional protein quality control system that operates via a distinctive mechanism. A protein chaperone system is identified that consists of proteins with poly-Asp/Glu sequence, and may have an important role in diseases characterized by protein aggregation.

Metastasizing cancer cells are able to withstand high levels of oxidative stress through mechanisms that are poorly understood. Here, we show that under various oxidative stress conditions, pancreatic cancer cells markedly expand NADPH and NADP + pools. This expansion is due to up-regulation of glucose-6-phosphate dehydrogenase (G6PD), which stimulates the cytoplasmic nicotinamide adenine dinucleotide kinase (NADK1) to produce NADP + while converting NADP + to NADPH. G6PD is activated by the transcription factor TAp73, which is, in turn, regulated by two pathways. Nuclear factor–erythroid 2 p45-related factor-2 suppresses expression of the ubiquitin ligase PIRH2, stabilizing the TAp73 protein. Checkpoint kinases 1/2 and E2F1 induce expression of the TAp73 gene. Levels of G6PD and its upstream activators are elevated in metastatic pancreatic cancer. Knocking down G6PD impedes pancreatic cancer metastasis, whereas forced G6PD expression promotes it. These findings reveal an intracellular network that maintains redox homeostasis through G6PD-mediated increase in de novo NADP + biosynthesis, which may be co-opted by tumor cells to enable metastasis.

Embryonic stem cells can propagate indefinitely in a pluripotent state, able to differentiate into all types of specialized cells when restored to the embryo. What sustains their pluripotency during propagation remains unclear. Here, we show that core pluripotency factors OCT4 and SOX2 suppress chaperone-mediated autophagy (CMA), a selective form of autophagy, until the initiation of differentiation. Low CMA activity promotes embryonic stem cell self-renewal, whereas its up-regulation enhances differentiation. CMA degrades isocitrate dehydrogenases IDH1 and IDH2 and reduces levels of intracellular α-ketoglutarate, an obligatory cofactor for various histone and DNA demethylases involved in pluripotency. These findings suggest that CMA mediates the effect of core pluripotency factors on metabolism, shaping the epigenetic landscape of stem cells and governing the balance between self-renewal and differentiation.

TCF-1 is a key transcription factor in progenitor exhausted CD8 T cells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single cell RNA-sequencing (scRNA-seq) and lineage tracing identified a TCF-1(+)Ly108(+)PD-1(+) CD8 T cell population early during chronic infection that seeds development of mature Tex cells. TCF-1 mediated the bifurcation between divergent fates, repressing development of terminal KLRG1(Hi) effectors while fostering KLRG1(Lo) Tex precursor cells, and PD-1 stabilized this TCF-1(+) Tex precursor cell pool. TCF-1 mediated a T-bet to Eomes transcription factor transition in Tex precursors by promoting Eomes expression and drove c-Myb expression that controlled Bcl-2 and survival. These data define a role for TCF-1 in early fate bifurcation driving Tex precursor cells, and also identify PD-1 as a protector of this early TCF-1 subset.

Frataxin is the protein that is down-regulated in Friedreich ataxia (FRDA), an autosomal recessive genetic disease caused by an intronic GAA repeat expansion in intron-1 of the FXN gene. The GAA repeats result in epigenetic silencing of the FXN gene and reduced expression of the cytosolic full-length frataxin (1−210) protein. Full length frataxin translocates to the mitochondria, leading to formation of mature frataxin (81–210) formed by cleavage of the mitochondrial targeting sequence at K-80 of the full-length protein. There are currently no approved treatments for FRDA, although experimental approaches involving up-regulation or replacement of mature frataxin protein through numerous approaches are being tested. Many of the pre-clinical studies of these experimental approaches are conducted in mouse and monkey models as well as in human cell lines. Consequently, well-validated antibodies are required for use in western blot analysis to determine whether levels of various forms of frataxin have been increased. Here we examined the specificity of five commercially available anti-frataxin antibodies and determined whether they detect mature frataxin in mouse heart tissue. Four protein standards of monkey, human, and mouse frataxin as well as mouse heart tissue were examined using polyacrylamide gel electrophoresis (PAGE) in combination with western blot analysis. One antibody failed to detect any of the frataxin standards or endogenous frataxin in mouse heart tissue. Three of the antibodies detected a protein in mouse heart tissue that ran slightly faster on PAGE (at 23.4 kDa) to that predicted for full-length frataxin (23.9 kDa). One antibody detected all four frataxin standards as well as endogenous mouse mature frataxin in mouse tissue. Significantly, this antibody, which will be useful for monitoring mature frataxin levels in monkey, human, and mouse tissues, did not detect a protein in mouse heart tissue at 23.4 kDa. Therefore, antibodies detecting the immunoreactive protein at 23.4 kDa could be misleading when testing for the up-regulation of frataxin in animal models.

RNA polymerase III (Pol III) is responsible for the production of small noncoding RNA species, including tRNAs and 5S rRNA. Pol III–dependent transcription is generally enhanced in transformed cells and tumors, but the underlying mechanisms remain not well-understood. It has been demonstrated that the BRF1 subunit of TFIIIB is essential for the accurate initiation of Pol III–dependent transcription. However, it is not known whether BRF1 undergoes ubiquitin modification and whether BRF1 ubiquitination regulates Pol III–dependent transcription. Here, we show that RNF12, a RING domain-containing ubiquitin E3 ligase, physically interacts with BRF1. Via direct interaction, RNF12 catalyzes Lys(27)- and Lys(33)-linked polyubiquitination of BRF1. Furthermore, RNF12 is able to negatively regulate Pol III–dependent transcription and cell proliferation via BRF1. These findings uncover a novel mechanism for the regulation of BRF1 and reveal RNF12 as an important regulator of Pol III–dependent transcription.

Summary The biological and functional heterogeneity between tumors—both across and within cancer types—poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.

MicroRNAs play an important role in CD8+ T cell differentiation and anti-viral immune responses. However, how microRNAs regulate CD8+ T cell memory formation remains poorly defined. Here, by microRNA profiling of CD8+ T cell during acute or chronic LCMV infection, we identified microRNAs that are specifically enriched in memory CD8+ T cells comparing to naïve, effector or exhausted CD8+ T cells. MiR-150, a microRNA that has been shown to be important in multiple biological processes, was identified as a potential memory CD8+ T cell biased microRNA. Functional interrogation of the role of miR-150 demonstrated that absence of miR-150 in CD8+ T cells enhanced memory CD8+ T cell differentiation, by both increasing KLRG-CD127+ memory precursors population during effector phase and enhancing CD127+CXCR3+ central memory phenotype long-term. Overexpression of miR-150 in CD8+ T cells, in contrast, negatively regulated CD8+ T cell memory formation. Furthermore, we found that miR-150 KO CD8+ T cells had higher expression of c-Myb comparing to miR-150 WT CD8+ T cells, a result observed using both in vitro CD8+ T cell differentiation and following infection with LCMV-Armstrong in vivo. Gene expression analysis implicated a relationship between the level of c-Myb expression and CD8+ T cell memory formation through potential anti-apoptotic molecules such as bcl-2. Thus, our studies identify a key role for miR-150 in CD8+ T cell memory and implicate a mechanism of CD8+ T cell memory effect by this microRNA through regulation of c-Myb.