HBO1 is required for the maintenance of leukaemia stem cells.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

Targeting Menin disrupts the KMT2A/B and polycomb balance to paradoxically activate bivalent genes.

Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR-Cas9 screens, we identify specific roles for MTF2-PRC2.1, PCGF1-PRC1.1 and Menin-KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin-KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.

Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death.

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples. SIGNIFICANCE: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587.

Inhibition of the CtBP complex and FBXO11 enhances MHC class II expression and anti-cancer immune responses.

There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.

The natural history of vascular and other complications in patients treated with nilotinib for chronic myeloid leukemia.

Although second-generation tyrosine kinase inhibitors (TKIs) show superiority in achieving deep molecular responses in chronic myeloid leukemia in chronic phase (CML-CP) compared with imatinib, the differing adverse effect (AE) profiles need consideration when deciding the best drug for individual patients. Long-term data from randomized trials of nilotinib demonstrate an increased risk of vascular AEs (VAEs) compared with other TKIs, although the natural history of these events in response to dose modifications or cessation has not been fully characterized. We retrospectively reviewed the incidence of nilotinib-associated AEs in 220 patients with CML-CP at 17 Australian institutions. Overall, AEs of any grade were reported in 95 patients (43%) and prompted nilotinib cessation in 46 (21%). VAEs occurred in 26 patients (12%), with an incidence of 4.1 events per 100 patient-years. Multivariate analysis identified age (P = .022) and dyslipidemia (P = .007) as independent variables for their development. There was 1 fatal first VAE, whereas the remaining patients either continued nilotinib (14 patients) or stopped it immediately (11 patients). Recurrent VAEs were associated with ongoing therapy in 7 of 14 who continued (with 2 fatal VAEs) vs 1 of 11 who discontinued (P = .04). Nineteen of the 23 evaluable patients surviving a VAE ultimately stopped nilotinib, of whom 14 received an alternative TKI. Dose reduction or cessation because of VAEs did not adversely affect maintenance of major molecular response. These findings demonstrate that in contrast to other AEs, VAEs are ideally managed with nilotinib cessation because of the increased risk of additional events with its ongoing use.

An Evolutionarily Conserved Function of Polycomb Silences the MHC Class I Antigen Presentation Pathway and Enables Immune Evasion in Cancer.

Loss of MHC class I (MHC-I) antigen presentation in cancer cells can elicit immunotherapy resistance. A genome-wide CRISPR/Cas9 screen identified an evolutionarily conserved function of polycomb repressive complex 2 (PRC2) that mediates coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP), promoting evasion of T cell-mediated immunity. MHC-I APP gene promoters in MHC-I low cancers harbor bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine-induced upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological MHC-I silencing highlights a conserved mechanism by which cancers arising from these primitive tissues exploit PRC2 activity to enable immune evasion.

Reference values for kaolin-activated thromboelastography in healthy children.

BACKGROUND: The hemostatic system of children changes with age and differs significantly from the hemostatic system of adults. Age-specific reference values are therefore required for most hemostatic variables. Thromboelastography (TEG) is a point-of-care coagulation test that may provide superior evaluation and management of coagulopathies after cardiac surgery, when large-dose unfractionated heparin is administered for cardiopulmonary bypass. In this study, we established reference values for kaolin-activated TEG in healthy children, to facilitate accurate interpretation of pediatric TEG results. METHODS: Kaolin-activated TEG was performed on 100 healthy children undergoing elective day surgery and 25 healthy adult volunteers. The following TEG variables were recorded: reaction time, coagulation time, alpha angle, maximum amplitude, percentage lysis 30 min after maximum amplitude was reached, and the coagulation index. Differences between age-groups were evaluated using analysis of variance. RESULTS: Age-specific reference values for kaolin-activated TEG in healthy children between 1 mo and 16 yr of age are presented. No significant differences between children and adults were observed. CONCLUSIONS: TEG results, from a particular clinical setting, must be compared to age-specific, as well as analyzer- and activator-specific, reference values to allow for correct interpretation of the results. Reference values provided here will be of use in acute clinical situations where a practical monitor of hemostasis is required.

The incidence and natural history of dasatinib complications in the treatment of chronic myeloid leukemia.

Dasatinib has shown superiority over imatinib in achieving molecular responses (MRs) in chronic phase chronic myeloid leukemia but with a different toxicity profile, which may impact its overall benefit. Reported toxicities include pleural effusions and pulmonary hypertension, and although the incidence of these events is well described, response to therapy and impact of dose modifications on toxicity has not been comprehensively characterized in a real-world setting. We retrospectively reviewed the incidence of dasatinib adverse events in 212 chronic phase chronic myeloid leukemia patients at 17 Australian institutions. Adverse events were reported in 116 patients (55%), most commonly pleural effusions (53 patients, 25%), which was the predominant cause of permanent drug cessation. Age and dose were risk factors for pleural effusion (P < .01 and .047, respectively). Recurrence rates were higher in those who remained on 100 mg compared with those who dose reduced (P = .041); however, recurrence still occurred at 50 mg. Patients who developed pleural effusions were more likely to have achieved MR4.5 after 6 months of dasatinib than those without effusions (P = .008). Pulmonary hypertension occurred in 5% of patients, frequently in association with pleural effusion, and was reversible upon dasatinib cessation in 6 of 7 patients. Dose reductions and temporary cessations had minimal impact on MR rates. Our observations suggest that by using the lowest effective dose in older patients to minimize the effusion risk, dose modification for cytopenias, and care with concomitant antiplatelet therapy, the necessity for permanent dasatinib cessation due to toxicity is likely to be minimal in immunologically competent patients.

Update and new approaches in the treatment of Castleman disease.

First described 60 years ago, Castleman disease comprises a rare and heterogeneous cluster of disorders, characterized by lymphadenopathy with unique histological features and associated with cytokine-driven constitutional symptoms and biochemical disturbances. Although unicentric Castleman disease is curable with complete surgical excision, its multicentric counterpart is a considerable therapeutic challenge. The recent development of biological agents, particularly monoclonal antibodies to interleukin-6 and its receptor, allow for more targeted disease-specific intervention that promises improved response rates and more durable disease control; however, further work is required to fill knowledge gaps in terms of underlying pathophysiology and to facilitate alternative treatment options for refractory cases.

Rituximab is associated with improved survival for aggressive B cell CNS lymphoma.

BACKGROUND: The optimal treatment strategy in patients with aggressive B cell central nervous system lymphoma suitable to receive intensive therapy is unknown. The benefit of incorporating rituximab in systemic therapy remains unclear. We performed a retrospective study examining the impact of rituximab in the context of concomitant therapies, including methotrexate, cytarabine, and radiotherapy, in patients treated with curative intent at 4 university teaching hospitals during 1996-2011. METHODS: A retrospective study of CNS lymphoma cases treated at the participating institutions was performed in accordance with institutional ethical guidelines. Patients were included if they received a diagnosis of primary diffuse large B cell lymphoma of the CNS, were HIV negative, and were treated with curative intent. RESULTS: One hundred twenty patients aged 21-81 years were identified. Rituximab recipients and nonrecipients were similar, except for rituximab recipients being more likely to have received a diagnosis after 2004. The median follow-up of surviving patients was 30 months. The 5-year overall survival was 46%. Univariate analysis revealed age ≤60 years, ECOG performance status ≤1, normal lactate dehydrogenase, diagnosis after 2004, and treatment with cytarabine and rituximab as predictive of favorable overall survival. Multivariate analysis identified age to be an independent predictor of overall survival, with a trend toward improved survival from the other variables that were significant in univariate analyses. CONCLUSIONS: In this retrospective analysis, the addition of rituximab to high-dose methotrexate-based chemotherapy in patients with aggressive B cell CNS lymphoma was associated with improved overall survival. Further studies are underway to prospectively validate these findings.