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The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.

A small-molecule inhibitor of the Mediator-associated kinases CDK8 and CDK19 inhibits growth of acute myeloid leukaemia (AML) cells and induces upregulation of super-enhancer-associated genes with tumour suppressor and lineage-controlling functions; Mediator kinase inhibition therefore represents a promising therapeutic approach for AML. Anti-leukaemic effect of Mediator kinase inhibition Super-enhancers are large enhancers densely bound by transcription factors, the Mediator complex and chromatin regulators, which drive high expression of genes implicated in cell identity and disease. Matthew Shair and colleagues find that a small molecule inhibitor of the Mediator-associated kinase CDK8 inhibits growth of acute myeloid leukaemia (AML) cells and induces upregulation of super-enhancer-associated genes linked to tumour suppressor and lineage-controlling functions. This enhancer upregulation is surprising given that a bromodomain BRD4 inhibitor suppresses AML cell growth yet downregulates the super-enhancer-associated genes. Therefore the AML cells seem to depend on a precise dosage of super-enhancer-associated gene expression and CDK8 inhibition. Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes 1 , 2 . BRD4 and CDK7 are positive regulators of SE-mediated transcription 3 , 4 , 5 . By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo , and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA , IRF8 , IRF1 and ETV6 (refs 6 , 7 , 8 ). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.

FBW7 acts by affecting apoptosis Loss of the tumour suppressor FBW7 is frequently observed in various types of human cancers, but its mechanism of action as a tumour suppressor remains unclear. Two groups demonstrate that in several cancer types, including ovarian cancer and T-cell leukaemias, the apoptosis regulator MCL1 is targeted for degradation by FBW7. Inuzuka et al . find that this mechanism can determine the response to drugs targeting BCL2 family apoptosis factors, and Wertz et al . show that it is activated during mitotic arrest and determines the response to anti-tubulin chemotherapeutics. Deletion or mutation of FBW7 in patients with cancer can therefore render tumours resistant to these therapies. This is one of two papers demonstrating that in several cancer types including ovarian cancer and T-cell leukaemias, the apoptosis regulator MCL1 is targeted for degradation by the FBW7 tumour suppressor. This study finds that this mechanism can determine the response to drugs targeting BCL2 family apoptosis factors. Deletion or mutation of FBW7 found in cancer patients therefore can render tumours resistant to these therapies. The effective use of targeted therapy is highly dependent on the identification of responder patient populations. Loss of FBW7 , which encodes a tumour-suppressor protein, is frequently found in various types of human cancer, including breast cancer, colon cancer 1 and T-cell acute lymphoblastic leukaemia (T-ALL) 2 . In line with these genomic data, engineered deletion of Fbw7 in mouse T cells results in T-ALL 3 , 4 , 5 , validating FBW7 as a T-ALL tumour suppressor. Determining the precise molecular mechanisms by which FBW7 exerts antitumour activity is an area of intensive investigation. These mechanisms are thought to relate in part to FBW7-mediated destruction of key proteins relevant to cancer, including Jun 6 , Myc 7 , cyclin E 8 and notch 1 (ref. 9 ), all of which have oncoprotein activity and are overexpressed in various human cancers, including leukaemia. In addition to accelerating cell growth 10 , overexpression of Jun, Myc or notch 1 can also induce programmed cell death 11 . Thus, considerable uncertainty surrounds how FBW7-deficient cells evade cell death in the setting of upregulated Jun, Myc and/or notch 1. Here we show that the E3 ubiquitin ligase SCF FBW7 (a SKP1–cullin-1–F-box complex that contains FBW7 as the F-box protein) governs cellular apoptosis by targeting MCL1, a pro-survival BCL2 family member, for ubiquitylation and destruction in a manner that depends on phosphorylation by glycogen synthase kinase 3. Human T-ALL cell lines showed a close relationship between FBW7 loss and MCL1 overexpression. Correspondingly, T-ALL cell lines with defective FBW7 are particularly sensitive to the multi-kinase inhibitor sorafenib but resistant to the BCL2 antagonist ABT-737. On the genetic level, FBW7 reconstitution or MCL1 depletion restores sensitivity to ABT-737, establishing MCL1 as a therapeutically relevant bypass survival mechanism that enables FBW7 -deficient cells to evade apoptosis. Therefore, our work provides insight into the molecular mechanism of direct tumour suppression by FBW7 and has implications for the targeted treatment of patients with FBW7 -deficient T-ALL.

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53 -wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53 -wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models. T- and NK-cell lymphomas (TCL) are a group of lymphoid malignancies characterized by poor prognosis, but the absence of appropriate pre-clinical models has hampered the development of effective therapies. Here the authors establish several pre-clinical models and identify vulnerabilities that could be further exploited to treat patients afflicted by these diseases.

Loss of PTEN expression, via homozygous or hemizygous deletion, is common in PIK3CA mutant ER + BC tumors. We assessed reduction of PTEN protein expression on AKT inhibitor capivasertib efficacy in PIK3CA altered tumors. In PIK3CA altered, PTEN protein high models, PI3Kα and AKT inhibition was effective, however ablation and partial PTEN expression reduction attenuated PI3Kαi but not AKTi efficacy, alone or combined with fulvestrant. Efficacy was FOXO3 dependent and associated with FOXM1 downregulation. FOXO3A deletion reduced response to capivasertib, and increased FOXM1 expression. Long term capivasertib exposure of ER+ BC cells upregulated FOXM1 expression. Downregulating FOXM1 expression reversed resistance to capivasertib, while FOXM1 overexpression reduced capivasertib efficacy. Collectively this suggests the AKT-FOXO3-FOXM1 axis plays a pivotal role in response to AKTi in ER+ breast cancer with PIK3CA mutations with and without expression of PTEN, that FOXO3 expression loss can mediate resistance, and that FOXM1 downregulation is a potential biomarker of response.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1 , as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo. A functional genomics approach in patient derived cell lines of Favorable Histology Wilms Tumor identifies suppression of TRIP13 through the nuclear export inhibitor, KPT-330, leads to decreased viability and synergy with doxorubicin.

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic ‘writers’ and ‘erasers’. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein–protein interactions of epigenetic ‘readers’, and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family. Histone mimics target BET bromodomains Small molecules that perturb chromatin proteins are an emerging focus of current biomedical research. Two groups reporting in this issue have targeted bromodomain-containing BET proteins that bind acetylated lysine residues during gene activation, arriving at cell-permeable small molecule compounds with similar structures based on fused triazole-diazepine rings. James Bradner and colleagues report the development of a compound named JQ1. The BET protein BRD4, with two bromodomains, is implicated in human squamous cell carcinoma. JQ1 inhibits the growth of BRD4-dependent tumours in mouse models. Alexander Tarakhovsky and colleagues' inhibitor, I-BET, is shown to interfere with the binding of certain BET family members to acetylated histones. It inhibits activation of pro-inflammatory genes in macrophages and has immunomodulatory activity in a mouse model of inflammatory disease. A new approach is used to target BET family bromodomains which are found in transcriptional regulators where they mediate the recognition of acetyl-lysine chromatin marks. Structural data reveal how the compound JQ1 binds to the bromodomain of BRD4. BRD4 has been implicated in a subtype of human squamous carcinomas, and JQ1 is found to inhibit the growth of BRD4 dependent tumours in mouse models.

The effective use of targeted therapy is highly dependent on the identification of responder patient populations. Loss of FBW7, which encodes a tumour-suppressor protein, is frequently found in various types of human cancer, including breast cancer, colon cancer and T-cell acute lymphoblastic leukaemia (T-ALL). In line with these genomic data, engineered deletion of Fbw7 in mouse T cells results in T-ALL, validating FBW7 as a T-ALL tumour suppressor. Determining the precise molecular mechanisms by which FBW7 exerts antitumour activity is an area of intensive investigation. These mechanisms are thought to relate in part to FBW7-mediated destruction of key proteins relevant to cancer, including Jun, Myc, cyclin E and notch 1 (ref. 9), all of which have oncoprotein activity and are overexpressed in various human cancers, including leukaemia. In addition to accelerating cell growth, overexpression of Jun, Myc or notch 1 can also induce programmed cell death. Thus, considerable uncertainty surrounds how FBW7-deficient cells evade cell death in the setting of upregulated Jun, Myc and/or notch 1. Here we show that the E3 ubiquitin ligase SCF(FBW7) (a SKP1-cullin-1-F-box complex that contains FBW7 as the F-box protein) governs cellular apoptosis by targeting MCL1, a pro-survival BCL2 family member, for ubiquitylation and destruction in a manner that depends on phosphorylation by glycogen synthase kinase 3. Human T-ALL cell lines showed a close relationship between FBW7 loss and MCL1 overexpression. Correspondingly, T-ALL cell lines with defective FBW7 are particularly sensitive to the multi-kinase inhibitor sorafenib but resistant to the BCL2 antagonist ABT-737. On the genetic level, FBW7 reconstitution or MCL1 depletion restores sensitivity to ABT-737, establishing MCL1 as a therapeutically relevant bypass survival mechanism that enables FBW7-deficient cells to evade apoptosis. Therefore, our work provides insight into the molecular mechanism of direct tumour suppression by FBW7 and has implications for the targeted treatment of patients with FBW7-deficient T-ALL.

There remains a need to identify new sensitive diagnostic and predictive blood-based platforms in lymphoma. We previously discovered a novel circulating microRNA (miRNA) signature in a Smurf2-deficient mouse model that spontaneously develops diffuse large B-cell lymphoma (DLBCL). Herein, we investigated this 10-miRNA signature (miR-15a, let-7c, let-7b, miR-27a, miR-10b, miR-18a, miR-497, miR-130a, miR24, and miR-155) in human lymphoma cell lines, mice engrafted with patient-derived xenografts (PDXs), and DLBCL patient serum samples leveraging systems biology analyses and droplet digital PCR (ddPCR) technology. Overall, 90% of the miRNAs were enriched in PDX DLBCL models and human lymphoma cell lines. Circulating miRNAs from the serum of 86 DLBCL patients were significantly increased compared with healthy controls and had similar patterns to the murine models. Strikingly, miRNAs were identified up to 27-fold higher levels in the serum of PDX-bearing mice and human patients compared with lymphoma cell lysates, suggesting a concentration of these factors over time within sera. Using cut-points from recursive partitioning analysis, we derived a 5-miRNA signature (let-7b, let-7c, miR-18a, miR-24, and miR-15a) with a classification rate of 91% for serum from patients with DLBCL versus normal controls. In addition, higher levels of circulating let-7b miRNA were associated with more advanced stage disease (i.e., III-IV vs. I-II) in DLBCL patients and higher levels of miR-27a and miR-24 were associated with MYC rearrangement. Taken together, circulating multi-miRNAs were readily detectable in pre-clinical cell line and human lymphoma models as well as in DLBCL patients where they appeared to distinguish clinico-pathologic subtypes and disease features.

The authors identify mutations in β subunits of G proteins that promote transformation and therapy resistance. Activating mutations in genes encoding G protein α (Gα) subunits occur in 4–5% of all human cancers 1 , but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a -deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.