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Cancer cells display DNA hypermethylation at specific CpG islands in comparison with their normal healthy counterparts, but the mechanism that drives this so-called CpG island methylator phenotype (CIMP) remains poorly understood. Here, we show that CpG island methylation in human T-cell acute lymphoblastic leukemia (T-ALL) mainly occurs at promoters of Polycomb Repressor Complex 2 (PRC2) target genes that are not expressed in normal or malignant T cells and that display a reciprocal association with H3K27me3 binding. In addition, we reveal that this aberrant methylation profile reflects the epigenetic history of T-ALL and is established already in preleukemic, self-renewing thymocytes that precede T-ALL development. Finally, we unexpectedly uncover that this age-related CpG island hypermethylation signature in T-ALL is completely resistant to the FDA-approved hypomethylating agent decitabine. Altogether, we provide conceptual evidence for the involvement of a preleukemic phase characterized by self-renewing thymocytes in the pathogenesis of human T-ALL.Significance:We developed a DNA methylation signature that reveals the epigenetic history of thymocytes during T-cell transformation. This human signature was recapitulated by murine self-renewing preleukemic thymocytes that build an age-related CpG island hypermethylation phenotype, providing conceptual evidence for the involvement of a preleukemic thymic phase in human T-cell leukemia.This article is highlighted in the In This Issue feature, p. 215

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The majority of cases of T-cell acute lymphoblastic leukemia (T-ALL) contain chromosomal abnormalities that drive overexpression of oncogenic transcription factors. However, whether these initiating oncogenes are required for leukemia maintenance is poorly understood. To address this, we developed a tetracycline-regulated mouse model of T-ALL driven by the oncogenic transcription factor Lmo2. This revealed that whilst thymus-resident pre-Leukemic Stem Cells (pre-LSCs) required continuous Lmo2 expression, the majority of leukemias relapsed despite Lmo2 withdrawal. Relapse was associated with a mature phenotype and frequent mutation or loss of tumor suppressor genes including Ikzf1 (Ikaros), with targeted deletion Ikzf1 being sufficient to transform Lmo2-dependent leukemias to Lmo2-independence. Moreover, we found that the related transcription factor TAL1 was dispensable in several human T-ALL cell lines that contain SIL-TAL1 chromosomal deletions driving its overexpression, indicating that evolution to oncogene independence can also occur in human T-ALL. Together these results indicate an evolution of oncogene addiction in murine and human T-ALL and show that loss of Ikaros is a mechanism that can promote self-renewal of T-ALL lymphoblasts in the absence of an initiating oncogenic transcription factor.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBF beta. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.

During the last decade, aberrant DNA methylation has been identified as a hallmark of human cancer and several studies have highlighted the potential of DNA methylation as a clinically or diagnostically relevant biomarker. In comparison to their normal healthy counterparts, cancer cells generally display DNA hypermethylation at specific CpG islands, but the actual mechanism that drives this so-called CpG island methylator phenotype (CIMP) remains poorly understood. To profile the DNA methylation landscape of human T-cell acute lymphoblastic leukemia (T-ALL), we analyzed 109 T-ALLs together with 10 stages of normal T cell development, which are considered the normal human counterparts of this disease, by 850 EPIC arrays. Here, we show that CpG islands are hypermethylated in all human T- ALLs compared to their normal counterparts. We designed a DNA methylation signature that can distinguish two types of T-ALL, with low or high levels of CpG island hypermethylation. This profile is dominated by CpGs in promoters of PRC2 target genes. T-ALLs with high levels of CpG island hypermethylation show low levels of H3K27me3 and vice versa, resulting in gene repression in both subtypes of T-ALL by different mechanisms. Furthermore, we found that aberrant CpG island hypermethylation shows a strong correlation with the epigenetic age of the leukemic T cells. By investigating the DNA methylation profile of two distinct mouse T-ALL models, the Lck-Cre Ptenfl/fland the CD2-Lmo2 transgenic mouse model, by Reduced Representation Bisulfite Sequencing, we could indeed recapitulate the DNA methylation features of the two human T-ALL subtypes in mice (Fig.1). The aggressive, fast-transforming Ptenfl/flmouse model displays low levels of CpG island hypermethylation, which correlated with human T-ALLs that have a shorter proliferative history and a worse prognosis. In contrast, murine CD2-Lmo2 T-ALLs have a longer disease latency and display a CpG island hypermethylation phenotype that is similar to human T-ALLs with a longer proliferative history. In CD2-Lmo2 mice, a pre-leukemic phase is present with self-renewing thymocytes. We elucidate that the CpG island methylation signature is gradually established in aging pre-leukemic thymocytes of 8, 16 and 24 weeks old CD2-Lmo2 mice (Fig.1). Of note, this hypermethylation phenotype is completely absent in age matched Lck-Cre Ptenfl/flmice that did not yet develop leukemia, suggesting that the proliferative history is responsible for aberrant CpG island DNA methylation observed in human T-ALL. Notably, this provides the first evidence that a pre-leukemic phase might be present in a large subset of human T-ALLs, and that epigenetic aberrations, either in the DNA methylation or histone methylation machinery are one of the first detectable alterations during T-ALL development. Finally, using patient derived xenografts (PDX), we show that DNA hypomethylation by the FDA-approved hypomethylating agent Decitabine is very effective in treating T-ALL. Gene expression profiling revealed that the anti-leukemic effect is exerted by down-regulation of the oncogenic MYC pathway. However, by profiling these PDX T-ALLs by EPIC arrays, we unexpectedly uncover that the age-related CpG island hypermethylation signature is completely resistant to Decitabine treatment. Altogether, our work demonstrates that DNA methylation reflects the epigenetic history of leukemic T cells and suggests that methylation-based subtypes of human T-ALL have followed a different trajectory towards T-cell transformation, possibly mediated by differences in the self-renewing capacity of the putative T-ALL cell-of-origin. Given that the concept of preleukemic thymocytes has only been reported in T-ALL mouse models so far, we here provide, for the first time, conceptual evidence that a pre-leukemic phase might also be involved in the pathogenesis of the human disease. Disclosures No relevant conflicts of interest to declare.

The majority of cases of T-cell acute lymphoblastic leukemia (T-ALL) contain chromosomal abnormalities that drive overexpression of oncogenic transcription factors. However, whether these initiating oncogenes are required for leukemia maintenance is poorly understood. To address this, we developed a tetracycline-regulated mouse model of T-ALL driven by the oncogenic transcription factor Lmo2. This revealed that whilst thymus-resident pre-Leukemic Stem Cells (pre-LSCs) required continuous Lmo2 expression, the majority of leukemias relapsed despite Lmo2 withdrawal. Relapse was associated with a mature phenotype and frequent mutation or loss of tumor suppressor genes including Ikzf1 (Ikaros), with targeted deletion Ikzf1 being sufficient to transform Lmo2-dependent leukemias to Lmo2-independence. Moreover, we found that the related transcription factor TAL1 was dispensable in several human T-ALL cell lines that contain SIL-TAL1 chromosomal deletions driving its overexpression, indicating that evolution to oncogene independence can also occur in human T-ALL. Together these results indicate an evolution of oncogene addiction in murine and human T-ALL and show that loss of Ikaros is a mechanism that can promote self-renewal of T-ALL lymphoblasts in the absence of an initiating oncogenic transcription factor.

Science advances 7(13), eabe1682 (2021). doi:10.1126/sciadv.abe1682, The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold���s termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics., Published by Assoc., Washington, DC [u.a.]

In cancer research, it remains challenging to functionally validate putative novel oncogenic drivers and to establish relevant preclinical models for evaluation of novel therapeutic strategies. Here, we describe an optimized and efficient pipeline for the generation of novel conditional overexpression mouse models in which putative oncogenes, along with an eGFP/Luciferase dual reporter, are expressed from the endogenous ROSA26 (R26) promoter. The efficiency of this approach was demonstrated by the generation and validation of novel R26 knock-in (KI) mice that allow conditional overexpression of Jarid2, Runx2, MN1 and a dominant negative allele of ETV6. As proof of concept, we confirm that MN1 overexpression in the hematopoietic lineage is sufficient to drive myeloid leukemia. In addition, we show that T-cell specific activation of MN1 in combination with loss of Pten increases tumour penetrance and stimulates the formation of Lyl1+ murine T-cell lymphoblastic leukemias or lymphomas (T-ALL/T-LBL). Finally, we demonstrate that these luciferase-positive murine AML and T-ALL/T-LBL cells are transplantable into immunocompromised mice allowing preclinical evaluation of novel anti-leukemic drugs in vivo.