TET2 lesions enhance the aggressiveness of CEBPA-mutant acute myeloid leukemia by rebalancing GATA2 expression.

The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated; CEBPADM) in acute myeloid leukemia (AML) with a combination of hypermorphic N-terminal mutations (CEBPANT), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations. The most frequently co-mutated genes in CEBPADM AML are GATA2 and TET2, however the molecular mechanisms underlying this co-mutational spectrum are incomplete. By combining transcriptomic and epigenomic analyses of CEBPA-TET2 co-mutated patients with models thereof, we identify GATA2 as a conserved target of the CEBPA-TET2 mutational axis, providing a rationale for the mutational spectra in CEBPADM AML. Elevated CEBPA levels, driven by CEBPANT, mediate recruitment of TET2 to the Gata2 distal hematopoietic enhancer thereby increasing Gata2 expression. Concurrent loss of TET2 in CEBPADM AML induces a competitive advantage by increasing Gata2 promoter methylation, thereby rebalancing GATA2 levels. Of clinical relevance, demethylating treatment of Cebpa-Tet2 co-mutated AML restores Gata2 levels and prolongs disease latency.

Arrayed CRISPR/Cas9 Screening for the Functional Validation of Cancer Genetic Dependencies.

CRISPR/Cas9 screening has revolutionized functional genomics in biomedical research and is a widely used approach for the identification of genetic dependencies in cancer cells. Here, we present an efficient and versatile protocol for the cloning of guide RNAs (gRNA) into lentiviral vectors, the production of lentiviral supernatants, and the transduction of target cells in a 96-well format. To assess the effect of gene knockouts on cellular fitness, we describe a competition-based cell proliferation assay using flow cytometry, enabling the screening of many genes at the same time in a fast and reproducible manner. This readout can be extended to any parameter that is accessible to flow-based measurements, such as protein expression and stability, differentiation, cell death, and others. In summary, this protocol allows to functionally assess the effect of a set of 50-300 gene knockouts on various cellular parameters within eight weeks. This protocol was validated in: Leukemia (2021), DOI: 10.1038/s41375-021-01169-6 Graphical abstract.

Downregulation of MTSS1 in acute myeloid leukemia is associated with a poor prognosis, chemotherapy resistance, and disease aggressiveness.

Despite recent approval of targeted drugs for acute myeloid leukemia (AML) therapy, chemotherapy with cytosine arabinoside and anthracyclines remains an important pillar of treatment. Both primary and secondary resistance are frequent and associated with poor survival, yet the underlying molecular mechanisms are incompletely understood. In previous work, we identified genes deregulated between diagnosis and relapse of AML, corresponding to therapy naïve and resistant states, respectively. Among them was MTSS1, whose downregulation is known to enhance aggressiveness of solid tumors. Here we show that low MTSS1 expression at diagnosis was associated with a poor prognosis in AML. MTSS1 expression was regulated by promoter methylation, and reduced by cytosine arabinoside and the anthracycline daunorubicin. Experimental downregulation of MTSS1 affected the expression of numerous genes. It induced the DNA damage response kinase WEE1, and rendered human AML cell lines more resistant to cytosine arabinoside, daunorubicin, and other anti-cancer drugs. Mtss1 knockdown in murine MLL-AF9-driven AML substantially decreased disease latency, and increased leukemic burden and ex vivo chemotherapy resistance. In summary, low MTSS1 expression represents a novel factor contributing to disease aggressiveness, therapy resistance, and poor outcome in AML.

Identification of gene targets of mutant C/EBPα reveals a critical role for MSI2 in CEBPA-mutated AML.

Mutations in the gene encoding the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) occur in 10-15% of acute myeloid leukemia (AML). Frameshifts in the CEBPA N-terminus resulting in exclusive expression of a truncated p30 isoform represent the most prevalent type of CEBPA mutations in AML. C/EBPα p30 interacts with the epigenetic machinery, but it is incompletely understood how p30-induced changes cause leukemogenesis. We hypothesized that critical effector genes in CEBPA-mutated AML are dependent on p30-mediated dysregulation of the epigenome. We mapped p30-associated regulatory elements (REs) by ATAC-seq and ChIP-seq in a myeloid progenitor cell model for p30-driven AML that enables inducible RNAi-mediated knockdown of p30. Concomitant p30-dependent changes in gene expression were measured by RNA-seq. Integrative analysis identified 117 p30-dependent REs associated with 33 strongly down-regulated genes upon p30-knockdown. CRISPR/Cas9-mediated mutational disruption of these genes revealed the RNA-binding protein MSI2 as a critical p30-target. MSI2 knockout in p30-driven murine AML cells and in the CEBPA-mutated human AML cell line KO-52 caused proliferation arrest and terminal myeloid differentiation, and delayed leukemia onset in vivo. In summary, this work presents a comprehensive dataset of p30-dependent effects on epigenetic regulation and gene expression and identifies MSI2 as an effector of the C/EBPα p30 oncoprotein.

Biomolecular condensation of NUP98 fusion proteins drives leukemogenic gene expression.

NUP98 fusion proteins cause leukemia via unknown molecular mechanisms. All NUP98 fusion proteins share an intrinsically disordered region (IDR) in the NUP98 N terminus, featuring repeats of phenylalanine-glycine (FG), and C-terminal fusion partners often function in gene control. We investigated whether mechanisms of oncogenic transformation by NUP98 fusion proteins are hardwired in their protein interactomes. Affinity purification coupled to mass spectrometry (MS) and confocal imaging of five NUP98 fusion proteins expressed in human leukemia cells revealed that shared interactors were enriched for proteins involved in biomolecular condensation and that they colocalized with NUP98 fusion proteins in nuclear puncta. We developed biotinylated isoxazole-mediated condensome MS (biCon-MS) to show that NUP98 fusion proteins alter the global composition of biomolecular condensates. An artificial FG-repeat-containing fusion protein phenocopied the nuclear localization patterns of NUP98 fusion proteins and their capability to drive oncogenic gene expression programs. Thus, we propose that IDR-containing fusion proteins combine biomolecular condensation with transcriptional control to induce cancer.

Regimen-dependent synergism and antagonism of treprostinil and vildagliptin in hematopoietic cell transplantation.

The cell dose in umbilical cord blood units is a major determinant for the outcome of hematopoietic cell transplantation. Prostaglandin analogs and dipeptidylpeptidase-4 (DPP4/CD26)-inhibitors enhance the ability of hematopoietic stem cells (HSCs) to reconstitute hematopoiesis. Here we explored the synergism between treprostinil, a stable prostaglandin agonist, and the DPP4/CD26-inhibitor vildagliptin. The combination of treprostinil and forskolin caused a modest but statistically significant increase in the surface levels of DPP4/CD26 on hematopoietic stem and progenitor cells (HSPCs) derived from murine bone and human cord blood. Their migration towards stromal cell-derived factor-1 (SDF-1/CXCL12) was enhanced, if they were pretreated with treprostinil and forskolin, and further augmented by vildagliptin. Administration of vildagliptin rescued 25% of lethally irradiated recipient mice injected with a limiting number of untreated HSPCs, but 90 to 100% of recipients injected with HSPCs preincubated with treprostinil and forskolin. The efficacy of vildagliptin surpassed that of treprostinil (60% rescue). Surprisingly, concomitant administration of vildagliptin and treprostinil resulted in poor survival of recipients indicating mutual antagonism, which was recapitulated when homing of and colony formation by HSPCs were assessed. These observations of regimen-dependent synergism and antagonism of treprostinil and vildagliptin are of translational relevance for the design of clinical trials. KEY MESSAGES: Pretreatment with treprostinil increases surface levels of DPP4/CD26 in HSPCs. Vildagliptin enhances in vitro migration of pretreated HSPCs. Vildagliptin enhances in vivo homing and engraftment of pretreated HSPCs. Unexpected mutual antagonism in vivo by concomitant administration of vildagliptin and treprostinil.

Gain-of-Function Effects of N-Terminal CEBPA Mutations in Acute Myeloid Leukemia.

Mutations in the CEBPA gene are present in 10-15% of acute myeloid leukemia (AML) patients. The most frequent type of mutations leads to the expression of an N-terminally truncated variant of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), termed p30. While initial reports proposed that p30 represents a dominant-negative version of the wild-type C/EBPα protein, other studies show that p30 retains the capacity to actively regulate gene expression. Recent global transcriptomic and epigenomic analyses have advanced the understanding of the distinct roles of the p30 isoform in leukemogenesis. This review outlines direct and indirect effects of the C/EBPα p30 variant on oncogenic transformation of hematopoietic progenitor cells and discusses how studies of N-terminal CEBPA mutations in AML can be extrapolated to identify novel gain-of-function features in oncoproteins that arise from recurrent truncating mutations in transcription factors.

CEBPA-mutated leukemia is sensitive to genetic and pharmacological targeting of the MLL1 complex.

The gene encoding the transcription factor C/EBPα is mutated in 10-15% of acute myeloid leukemia (AML) patients. N-terminal CEBPA mutations cause ablation of full-length C/EBPα without affecting the expression of a shorter oncogenic isoform, termed p30. The mechanistic basis of p30-induced leukemogenesis is incompletely understood. Here, we demonstrate that the MLL1 histone-methyltransferase complex represents a critical actionable vulnerability in CEBPA-mutated AML. Oncogenic C/EBPα p30 and MLL1 show global co-localization on chromatin and p30 exhibits robust physical interaction with the MLL1 complex. CRISPR/Cas9-mediated mutagenesis of MLL1 results in proliferation arrest and myeloid differentiation in C/EBPα p30-expressing cells. In line, CEBPA-mutated hematopoietic progenitor cells are hypersensitive to pharmacological targeting of the MLL1 complex. Inhibitor treatment impairs proliferation and restores myeloid differentiation potential in mouse and human AML cells with CEBPA mutations. Finally, we identify the transcription factor GATA2 as a direct critical target of the p30-MLL1 interaction. Altogether, we show that C/EBPα p30 requires the MLL1 complex to regulate oncogenic gene expression and that CEBPA-mutated AML is hypersensitive to perturbation of the MLL1 complex. These findings identify the MLL1 complex as a potential therapeutic target in AML with CEBPA mutations.

Pharmacologic inhibition of STAT5 in acute myeloid leukemia.

The transcription factor STAT5 is an essential downstream mediator of many tyrosine kinases (TKs), particularly in hematopoietic cancers. STAT5 is activated by FLT3-ITD, which is a constitutively active TK driving the pathogenesis of acute myeloid leukemia (AML). Since STAT5 is a critical mediator of diverse malignant properties of AML cells, direct targeting of STAT5 is of significant clinical value. Here, we describe the development and preclinical evaluation of a novel, potent STAT5 SH2 domain inhibitor, AC-4-130, which can efficiently block pathological levels of STAT5 activity in AML. AC-4-130 directly binds to STAT5 and disrupts STAT5 activation, dimerization, nuclear translocation, and STAT5-dependent gene transcription. Notably, AC-4-130 substantially impaired the proliferation and clonogenic growth of human AML cell lines and primary FLT3-ITD+ AML patient cells in vitro and in vivo. Furthermore, AC-4-130 synergistically increased the cytotoxicity of the JAK1/2 inhibitor Ruxolitinib and the p300/pCAF inhibitor Garcinol. Overall, the synergistic effects of AC-4-130 with TK inhibitors (TKIs) as well as emerging treatment strategies provide new therapeutic opportunities for leukemia and potentially other cancers.

Chemically Induced Degradation of the Oncogenic Transcription Factor BCL6.

The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.