Fusion proteins involving Nucleoporin 98 (NUP98) are recurrently found in acute myeloid leukemia (AML) and are associated with poor prognosis. Lack of mechanistic insight into NUP98-fusion–dependent oncogenic transformation has so far precluded the development of rational targeted therapies. We reasoned that different NUP98-fusion proteins deregulate a common set of transcriptional targets that might be exploitable for therapy. To decipher transcriptional programs controlled by diverse NUP98-fusion proteins, we developed mouse models for regulatable expression of NUP98/NSD1, NUP98/JARID1A, and NUP98/DDX10. By integrating chromatin occupancy profiles of NUP98-fusion proteins with transcriptome profiling upon acute fusion protein inactivation in vivo, we defined the core set of direct transcriptional targets of NUP98-fusion proteins. Among those, CDK6 was highly expressed in murine and human AML samples. Loss of CDK6 severely attenuated NUP98-fusion–driven leukemogenesis, and NUP98-fusion AML was sensitive to pharmacologic CDK6 inhibition in vitro and in vivo. These findings identify CDK6 as a conserved, critical direct target of NUP98-fusion proteins, proposing CDK4/CDK6 inhibitors as a new rational treatment option for AML patients with NUP98-fusions.

The non-redundant histone methyltransferase SETD2 (SET domain containing 2; KMT3A) is responsible for tri-methylation of lysine 36 on histone H3 (H3K36me3). Presence of the H3K36me3 histone mark across the genome has been correlated with transcriptional activation and elongation, but also with the regulation of DNA mismatch repair, homologous recombination and alternative splicing. The role of SETD2 and the H3K36me3 histone mark in cancer is controversial. SETD2 is lost or mutated in various cancers, supporting a tumor suppressive role of the protein. Alterations in the SETD2 gene are also present in leukemia patients, where they are associated with aggressive disease and relapse. In line, heterozygous SETD2 loss caused chemotherapy resistance in leukemia cell lines and mouse models. In contrast, other studies indicate that SETD2 is critically required for the proliferation of leukemia cells. Thus, although studies of SETD2-dependent processes in cancer have contributed to a better understanding of the SETD2–H3K36me3 axis, many open questions remain regarding its specific role in leukemia. Here, we review the current literature about critical functions of SETD2 in the context of hematopoietic malignancies.

MLL-fusions represent a large group of leukemia drivers, whose diversity originates from the vast molecular heterogeneity of C-terminal fusion partners of MLL. While studies of selected MLL-fusions have revealed critical molecular pathways, unifying mechanisms across all MLL-fusions remain poorly understood. We present the first comprehensive survey of protein–protein interactions of seven distantly related MLL-fusion proteins. Functional investigation of 128 conserved MLL-fusion-interactors identifies a specific role for the lysine methyltransferase SETD2 in MLL-leukemia. SETD2 loss causes growth arrest and differentiation of AML cells, and leads to increased DNA damage. In addition to its role in H3K36 tri-methylation, SETD2 is required to maintain high H3K79 di-methylation and MLL-AF9-binding to critical target genes, such as Hoxa9. SETD2 loss synergizes with pharmacologic inhibition of the H3K79 methyltransferase DOT1L to induce DNA damage, growth arrest, differentiation, and apoptosis. These results uncover a dependency for SETD2 during MLL-leukemogenesis, revealing a novel actionable vulnerability in this disease.

The BCL-2 inhibitor Venetoclax is a promising agent for the treatment of acute myeloid leukemia (AML). However, many patients are refractory to Venetoclax, and resistance develops quickly. ATP-binding cassette (ABC) transporters mediate chemotherapy resistance but their role in modulating the activity of targeted small-molecule inhibitors is unclear. Using CRISPR/Cas9 screening, we find that loss of ABCC1 strongly increases the sensitivity of AML cells to Venetoclax. Genetic and pharmacologic ABCC1 inactivation potentiates the anti-leukemic effects of BCL-2 inhibitors and efficiently re-sensitizes Venetoclax-resistant leukemia cells. Conversely, ABCC1 overexpression induces resistance to BCL-2 inhibitors by reducing intracellular drug levels, and high ABCC1 levels predicts poor response to Venetoclax therapy in patients. Consistent with ABCC1-specific export of glutathionylated substrates, inhibition of glutathione metabolism increases the potency of BCL-2 inhibitors. These results identify ABCC1 and glutathione metabolism as mechanisms limiting efficacy of BCL-2 inhibitors, which may pave the way to development of more effective therapies.

Histone methyltransferases (HMTs) have attracted considerable attention as potential targets for pharmaceutical intervention in various malignant diseases. These enzymes are known for introducing methyl marks at specific locations of histone proteins, creating a complex system that regulates epigenetic control of gene expression and cell differentiation. Here, we describe the identification of first-generation cell-permeable non-nucleoside type inhibitors of SETD2, the only mammalian HMT that is able to tri-methylate the K36 residue of histone H3. By generating the epigenetic mark H3K36me3, SETD2 is involved in the progression of acute myeloid leukemia. We developed a structure-based virtual screening protocol that was first validated in retrospective studies. Next, prospective screening was performed on a large library of commercially available compounds. Experimental validation of 22 virtual hits led to the discovery of three compounds that showed dose-dependent inhibition of the enzymatic activity of SETD2. Compound C13 effectively blocked the proliferation of two acute myeloid leukemia (AML) cell lines with MLL rearrangements and led to decreased H3K36me3 levels, prioritizing this chemotype as a viable chemical starting point for drug discovery projects.

Hypophosphatasia (HPP) is an inherited disorder that affects bone and tooth mineralization, among other body systems. HPP is caused by pathogenic variants in the alkaline phosphatase-liver (ALPL) gene, which encodes tissue nonspecific alkaline phosphatase. One major challenge in diagnosing HPP is interpreting variant of uncertain significance (VUS), which can create uncertainty for patients and healthcare professionals, leading to delays in diagnosis and treatment or incorrect diagnoses. Since February 2021, the ALPLgene variant consortium has reclassified 100 VUS using adjusted American College of Medical Genetics/Association for Molecular Pathology criteria. Of these, 11 were reclassified as pathogenic, 62 as likely pathogenic, three as likely benign, one as benign, and 23 remained as VUS; out of 100 variants, there were 81 missense, eight frameshifts, four in-frame deletions, two intronic, two synonymous, two nonsense, and one splice site variant. In vitrofunctional testing of the variant’s residual enzymatic activity and its dominant negative effect (DNE) was required for 40 variants.Out of 40 variants tested, 72% showed <30% residual activity and were reclassified as pathogenic or likely pathogenic. Among these, 48% exhibited a DNE. All variants with ˂15% residual activity showed a DNE, while those with over 20% did not. In terms of domains, all tested variants in the active site domain and 75% of those in the homodimeric interface showed <30% residual activity. The consortium’s reclassification of 100 ALPLvariants has resulted in a 21% increase in the number of variants in the Johannes Kepler University ALPLgene variant database. Furthermore, 136 new genotypes with 118 associated phenotypes, including asymptomatic states, have been added. These expanded resources will help improve genetic counseling for patients and families affected by ALPLvariants and enable faster diagnosis and medical decision-making.