The menin inhibitor revumenib in KMT2A-rearranged or NPM1-mutant leukaemia.

Targeting critical epigenetic regulators reverses aberrant transcription in cancer, thereby restoring normal tissue function1-3. The interaction of menin with lysine methyltransferase 2A (KMT2A), an epigenetic regulator, is a dependence in acute leukaemia caused by either rearrangement of KMT2A or mutation of the nucleophosmin 1 gene (NPM1)4-6. KMT2A rearrangements occur in up to 10% of acute leukaemias and have an adverse prognosis, whereas NPM1 mutations occur in up to 30%, forming the most common genetic alteration in acute myeloid leukaemia7,8. Here, we describe the results of the first-in-human phase 1 clinical trial investigating revumenib (SNDX-5613), a potent and selective oral inhibitor of the menin-KMT2A interaction, in patients with relapsed or refractory acute leukaemia (ClinicalTrials.gov, NCT04065399). We show that therapy with revumenib was associated with a low frequency of grade 3 or higher treatment-related adverse events and a 30% rate of complete remission or complete remission with partial haematologic recovery (CR/CRh) in the efficacy analysis population. Asymptomatic prolongation of the QT interval on electrocardiography was identified as the only dose-limiting toxicity. Remissions occurred in leukaemias refractory to multiple previous lines of therapy. We demonstrate clearance of residual disease using sensitive clinical assays and identify hallmarks of differentiation into normal haematopoietic cells, including differentiation syndrome. These data establish menin inhibition as a therapeutic strategy for susceptible acute leukaemia subtypes.

Mezigdomide is effective alone and in combination with menin inhibition in preclinical models of KMT2A-r and NPM1c AML.

ABSTRACT: Small molecules that target the menin-KMT2A protein-protein interaction (menin inhibitors) have recently entered clinical trials in lysine methyltransferase 2A (KMT2A or MLL1)-rearranged (KMT2A-r) and nucleophosmin-mutant (NPM1c) acute myeloid leukemia (AML) and are demonstrating encouraging results. However, rationally chosen combination therapy is needed to improve responses and prevent resistance. We have previously identified IKZF1/IKAROS as a target in KMT2A-r AML and shown in preclinical models that IKAROS protein degradation with lenalidomide or iberdomide has modest single-agent activity yet can synergize with menin inhibitors. Recently, the novel IKAROS degrader mezigdomide was developed with greatly enhanced IKAROS protein degradation. In this study, we show that mezigdomide has increased preclinical activity in vitro as a single-agent in KMT2A-r and NPM1c AML cell lines, including sensitivity in cell lines resistant to lenalidomide and iberdomide. Further, we demonstrate that mezigdomide has the greatest capacity to synergize with and induce apoptosis in combination with menin inhibitors, including in MEN1 mutant models. We show that the superior activity of mezigdomide compared with lenalidomide or iberdomide is due to its increased depth, rate, and duration of IKAROS protein degradation. Single-agent mezigdomide was efficacious in 5 patient-derived xenograft models of KMT2A-r and 1 NPM1c AML. The combination of mezigdomide with the menin inhibitor VTP-50469 increased survival and prevented and overcame MEN1 mutations that mediate resistance in patients receiving menin inhibitor monotherapy. These results support prioritization of mezigdomide for early phase clinical trials in KMT2A-r and NPM1c AML, either as a single agent or in combination with menin inhibitors.

Preclinical efficacy of potent and selective menin-KMT2A inhibitor JNJ-75276617 in KMT2A- and NPM1-altered leukemias.

The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) AML cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent anti-proliferative activity across several AML and ALL cell lines and patient samples harboring KMT2A- or NPM1-alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent anti-proliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A co-crystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory (R/R) acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903).

Functional TRIM24 degrader via conjugation of ineffectual bromodomain and VHL ligands.

The addressable pocket of a protein is often not functionally relevant in disease. This is true for the multidomain, bromodomain-containing transcriptional regulator TRIM24. TRIM24 has been posited as a dependency in numerous cancers, yet potent and selective ligands for the TRIM24 bromodomain do not exert effective anti-proliferative responses. We therefore repositioned these probes as targeting features for heterobifunctional protein degraders. Recruitment of the VHL E3 ubiquitin ligase by dTRIM24 elicits potent and selective degradation of TRIM24. Using dTRIM24 to probe TRIM24 function, we characterize the dynamic genome-wide consequences of TRIM24 loss on chromatin localization and gene control. Further, we identify TRIM24 as a novel dependency in acute leukemia. Pairwise study of TRIM24 degradation versus bromodomain inhibition reveals enhanced anti-proliferative response from degradation. We offer dTRIM24 as a chemical probe of an emerging cancer dependency, and establish a path forward for numerous selective yet ineffectual ligands for proteins of therapeutic interest.

The dTAG system for immediate and target-specific protein degradation.

Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.

Discovery of selective small-molecule HDAC6 inhibitor for overcoming proteasome inhibitor resistance in multiple myeloma.

Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM.

Complementary genomic approaches highlight the PI3K/mTOR pathway as a common vulnerability in osteosarcoma.

Osteosarcoma is the most common primary bone tumor, yet there have been no substantial advances in treatment or survival in three decades. We examined 59 tumor/normal pairs by whole-exome, whole-genome, and RNA-sequencing. Only the TP53 gene was mutated at significant frequency across all samples. The mean nonsilent somatic mutation rate was 1.2 mutations per megabase, and there was a median of 230 somatic rearrangements per tumor. Complex chains of rearrangements and localized hypermutation were detected in almost all cases. Given the intertumor heterogeneity, the extent of genomic instability, and the difficulty in acquiring a large sample size in a rare tumor, we used several methods to identify genomic events contributing to osteosarcoma survival. Pathway analysis, a heuristic analytic algorithm, a comparative oncology approach, and an shRNA screen converged on the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway as a central vulnerability for therapeutic exploitation in osteosarcoma. Osteosarcoma cell lines are responsive to pharmacologic and genetic inhibition of the PI3K/mTOR pathway both in vitro and in vivo.

Pharmacological targeting of the cancer epigenome.

Epigenetic dysregulation is increasingly appreciated as a hallmark of cancer, including disease initiation, maintenance and therapy resistance. As a result, there have been advances in the development and evaluation of epigenetic therapies for cancer, revealing substantial promise but also challenges. Three epigenetic inhibitor classes are approved in the USA, and many more are currently undergoing clinical investigation. In this Review, we discuss recent developments for each epigenetic drug class and their implications for therapy, as well as highlight new insights into the role of epigenetics in cancer.

Inherent genome instability underlies trisomy 21-associated myeloid malignancies.

Constitutional trisomy 21 (T21) is a state of aneuploidy associated with high incidence of childhood acute myeloid leukemia (AML). T21-associated AML is preceded by transient abnormal myelopoiesis (TAM), which is triggered by truncating mutations in GATA1 generating a short GATA1 isoform (GATA1s). T21-associated AML emerges due to secondary mutations in hematopoietic clones bearing GATA1s. Since aneuploidy generally impairs cellular fitness, the paradoxically elevated risk of myeloid malignancy in T21 is not fully understood. We hypothesized that individuals with T21 bear inherent genome instability in hematopoietic lineages that promotes leukemogenic mutations driving the genesis of TAM and AML. We found that individuals with T21 show increased chromosomal copy number variations (CNVs) compared to euploid individuals, suggesting that genome instability could be underlying predisposition to TAM and AML. Acquisition of GATA1s enforces myeloid skewing and maintenance of the hematopoietic progenitor state independently of T21; however, GATA1s in T21 hematopoietic progenitor cells (HPCs) further augments genome instability. Increased dosage of the chromosome 21 (chr21) gene DYRK1A impairs homology-directed DNA repair as a mechanism of elevated mutagenesis. These results posit a model wherein inherent genome instability in T21 drives myeloid malignancy in concert with GATA1s mutations.

Unleashing Cell-Intrinsic Inflammation as a Strategy to Kill AML Blasts.

Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as cell-intrinsic, self-directed immunotherapy. To this end, we applied genome-wide screens to discover genetic vulnerabilities in acute myeloid leukemia (AML) cells implicated in inflammatory pathways. We identified the immune modulator IRF2BP2 as a selective AML dependency. We validated AML cell dependency on IRF2BP2 with genetic and protein degradation approaches in vitro and genetically in vivo. Chromatin and global gene-expression studies demonstrated that IRF2BP2 represses IL1ß/TNFα signaling via NFκB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival. SIGNIFICANCE: This study exploits inflammatory programs inherent to AML blasts to identify genetic vulnerabilities in this disease. In doing so, we determined that AML cells are dependent on the transcriptional repressive activity of IRF2BP2 for their survival, revealing cell-intrinsic inflammation as a mechanism priming leukemic blasts for regulated cell death. See related commentary by Puissant and Medyouf, p. 1617. This article is highlighted in the In This Issue feature, p. 1599.

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest.

BACKGROUND: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. RESULTS: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. CONCLUSIONS: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.

DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7.

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.

A role for PP1 in the Cdc2/Cyclin B-mediated positive feedback activation of Cdc25.

The Cdc25 phosphatase promotes entry into mitosis through the removal of inhibitory phosphorylations on the Cdc2 subunit of the Cdc2/CyclinB complex. During interphase, or after DNA damage, Cdc25 is suppressed by phosphorylation at Ser287 (Xenopus numbering; Ser216 of human Cdc25C) and subsequent binding of the small acidic protein, 14-3-3. As reported recently, at the time of mitotic entry, 14-3-3 protein is removed from Cdc25 and S287 is dephosphorylated by protein phosphatase 1 (PP1). After the initial activation of Cdc25 and consequent derepression of Cdc2/CyclinB, Cdc25 is further activated through a Cdc2-catalyzed positive feedback loop. Although the existence of such a loop has been appreciated for some time, the molecular mechanism for this activation has not been described. We report here that phosphorylation of S285 by Cdc2 greatly enhances recruitment of PP1 to Cdc25, thereby accelerating S287 dephosphorylation and mitotic entry. Moreover, we show that two other previously reported sites of Cdc2-catalyzed phosphorylation on Cdc25 are required for maximal biological activity of Cdc25, but they do not contribute to PP1 regulation and do not act solely through controlling S287 phosphorylation. Therefore, multiple mechanisms, including enhanced recruitment of PP1, are used to promote full activation of Cdc25 at the time of mitotic entry.

A Menin-MLL Inhibitor Induces Specific Chromatin Changes and Eradicates Disease in Models of MLL-Rearranged Leukemia.

Inhibition of the Menin (MEN1) and MLL (MLL1, KMT2A) interaction is a potential therapeutic strategy for MLL-rearranged (MLL-r) leukemia. Structure-based design yielded the potent, highly selective, and orally bioavailable small-molecule inhibitor VTP50469. Cell lines carrying MLL rearrangements were selectively responsive to VTP50469. VTP50469 displaced Menin from protein complexes and inhibited chromatin occupancy of MLL at select genes. Loss of MLL binding led to changes in gene expression, differentiation, and apoptosis. Patient-derived xenograft (PDX) models derived from patients with either MLL-r acute myeloid leukemia or MLL-r acute lymphoblastic leukemia (ALL) showed dramatic reductions of leukemia burden when treated with VTP50469. Multiple mice engrafted with MLL-r ALL remained disease free for more than 1 year after treatment. These data support rapid translation of this approach to clinical trials.

Correction to 'Walking with wider steps changes foot placement control, increases kinematic variability and does not improve linear stability'.

[This corrects the article DOI: 10.1098/rsos.160627.].

Structure-Guided Design and Development of Potent and Selective Dual Bromodomain 4 (BRD4)/Polo-like Kinase 1 (PLK1) Inhibitors.

The simultaneous inhibition of polo-like kinase 1 (PLK1) and BRD4 bromodomain by a single molecule could lead to the development of an effective therapeutic strategy for a variety of diseases in which PLK1 and BRD4 are implicated. Compound 23 has been found to be a potent dual kinase-bromodomain inhibitor (BRD4-BD1 IC50 = 28 nM, PLK1 IC50 = 40 nM). Compound 6 was found to be the most selective PLK1 inhibitor over BRD4 in our series (BRD4-BD1 IC50 = 2579 nM, PLK1 IC50 = 9.9 nM). Molecular docking studies with 23 and BRD4-BD1/PLK1 as well as with 6 corroborate the biochemical assay results.

YAP1-Mediated Suppression of USP31 Enhances NFκB Activity to Promote Sarcomagenesis.

To date, no consistent oncogenic driver mutations have been identified in most adult soft tissue sarcomas; these tumors are thus generally insensitive to existing targeted therapies. Here we investigated alternate mechanisms underlying sarcomagenesis to identify potential therapeutic interventions. Undifferentiated pleomorphic sarcoma (UPS) is an aggressive tumor frequently found in skeletal muscle where deregulation of the Hippo pathway and aberrant stabilization of its transcriptional effector yes-associated protein 1 (YAP1) increases proliferation and tumorigenesis. However, the downstream mechanisms driving this deregulation are incompletely understood. Using autochthonous mouse models and whole genome analyses, we found that YAP1 was constitutively active in some sarcomas due to epigenetic silencing of its inhibitor angiomotin (AMOT). Epigenetic modulators vorinostat and JQ1 restored AMOT expression and wild-type Hippo pathway signaling, which induced a muscle differentiation program and inhibited sarcomagenesis. YAP1 promoted sarcomagenesis by inhibiting expression of ubiquitin-specific peptidase 31 (USP31), a newly identified upstream negative regulator of NFκB signaling. Combined treatment with epigenetic modulators effectively restored USP31 expression, resulting in decreased NFκB activity. Our findings highlight a key underlying molecular mechanism in UPS and demonstrate the potential impact of an epigenetic approach to sarcoma treatment.Significance: A new link between Hippo pathway signaling, NFκB, and epigenetic reprogramming is highlighted and has the potential for therapeutic intervention in soft tissue sarcomas. Cancer Res; 78(10); 2705-20. ©2018 AACR.

Walking with wider steps changes foot placement control, increases kinematic variability and does not improve linear stability.

Walking humans respond to pulls or pushes on their upper body by changing where they place their foot on the next step. Usually, they place their foot further along the direction of the upper body perturbation. Here, we examine how this foot placement response is affected by the average step width during walking. We performed experiments with humans walking on a treadmill, both normally and at five different prescribed step widths. We prescribed step widths by requiring subjects to step on lines drawn on the treadmill belt. We inferred a linear model between the torso marker state at mid-stance and the next foot position. The coefficients in this linear model (which are analogous to feedback gains for foot placement) changed with increasing step width as follows. The sideways foot placement response to a given sideways torso deviation decreased. The fore-aft foot placement response to a given fore-aft torso deviation also decreased. Coupling between fore-aft foot placement and sideways torso deviations increased. These changes in foot placement feedback gains did not significantly affect walking stability as quantified by Floquet multipliers (which estimate how quickly the system corrects a small perturbation), despite increasing foot placement variance and upper body motion variance (kinematic variability).

PRMT1-Mediated Translation Regulation Is a Crucial Vulnerability of Cancer.

Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control. In particular, loss of Prmt1 led to a decrease in arginine methylation of the translation initiation complex, thereby disrupting its assembly and inhibiting translation. p53/Rb-null cells were sensitive to p53-induced translation stress, and analysis of human cancer cell line data from Project Achilles further revealed that Prmt1 and translation-associated pathways converged on the same functional networks. We propose that targeted therapy against Prmt1 and its associated translation-related pathways offer a mechanistic rationale for treatment of osteosarcomas and other cancers that exhibit dependencies on translation stress response. Cancer Res; 77(17); 4613-25. ©2017 AACR.