BRD9 degraders as chemosensitizers in acute leukemia and multiple myeloma.

Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.

Essential role of the histone lysine demethylase KDM4A in the biology of malignant pleural mesothelioma (MPM).

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM. METHODS: KDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways. RESULTS: Levels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth. CONCLUSIONS: The results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.

Inhibitors of the Transcription Factor STAT3 Decrease Growth and Induce Immune Response Genes in Models of Malignant Pleural Mesothelioma (MPM).

Malignant pleural mesothelioma (MPM) is an aggressive cancer defined by loss-of-function mutations with few therapeutic options. We examined the contribution of the transcription factor Signal transducer and activator of transcription 3 (STAT3) to cell growth and gene expression in preclinical models of MPM. STAT3 is activated in a variety of tumors and is thought to be required for the maintenance of cancer stem cells. Targeting STAT3 using specific small hairpin RNAs (shRNAs) or with the pharmacologic inhibitors atovaquone or pyrimethamine efficiently reduced cell growth in established cell lines and primary-derived lines while showing minimal effects in nontransformed LP9 mesothelial cells. Moreover, atovaquone significantly reduced viability and tumor growth in microfluidic cultures of primary MPM as well as in an in vivo xenotransplant model. Biological changes were linked to modulation of gene expression associated with STAT3 signaling, including cell cycle progression and altered p53 response. Reflecting the role of STAT3 in inducing localized immune suppression, using both atovaquone and pyrimethamine resulted in the modulation of immunoregulatory genes predicted to enhance an immune response, including upregulation of ICOSLG (Inducible T-Cell Costimulator Ligand or B7H2). Thus, our data strongly support a role for STAT3 inhibitors as anti-MPM therapeutics.

Effects of the multi-kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia.

Recently, several targeted agents have been developed for specific subsets of patients with acute myeloid leukaemia (AML), including midostaurin, the first FDA-approved FLT3 inhibitor for newly diagnosed patients with FLT3 mutations. However, in the initial Phase I/II clinical trials, some patients without FLT3 mutations had transient responses to midostaurin, suggesting that this multi-targeted kinase inhibitor might benefit AML patients more broadly. Here, we demonstrate submicromolar efficacy of midostaurin in vitro and efficacy in vivo against wild-type (wt) FLT3-expressing AML cell lines and primary cells, and we compare its effectiveness with that of other FLT3 inhibitors currently in clinical trials. Midostaurin was found to synergize with standard chemotherapeutic drugs and some targeted agents against AML cells without mutations in FLT3. The mechanism may involve, in part, the unique kinase profile of midostaurin that includes proteins implicated in AML transformation, such as SYK or KIT, or inhibition of ERK pathway or proviability signalling. Our findings support further investigation of midostaurin as a chemosensitizing agent in AML patients without FLT3 mutations.

The combination of FLT3 and SYK kinase inhibitors is toxic to leukaemia cells with CBL mutations.

Mutations in the E3 ubiquitin ligase CBL, found in several myeloid neoplasms, lead to decreased ubiquitin ligase activity. In murine systems, these mutations are associated with cytokine-independent proliferation, thought to result from the activation of hematopoietic growth receptors, including FLT3 and KIT. Using cell lines and primary patient cells, we compared the activity of a panel of FLT3 inhibitors currently being used or tested in AML patients and also evaluated the effects of inhibition of the non-receptor tyrosine kinase, SYK. We show that FLT3 inhibitors ranging from promiscuous to highly targeted are potent inhibitors of growth of leukaemia cells expressing mutant CBL in vitro, and we demonstrate in vivo efficacy of midostaurin using mouse models of mutant CBL. Potentiation of effects of targeted FLT3 inhibition by SYK inhibition has been demonstrated in models of mutant FLT3-positive AML and AML characterized by hyperactivated SYK. Here, we show that targeted SYK inhibition similarly enhances the effects of midostaurin and other FLT3 inhibitors against mutant CBL-positive leukaemia. Taken together, our results support the notion that mutant CBL-expressing myeloid leukaemias are highly sensitive to available FLT3 inhibitors and that this effect can be significantly augmented by optimum inhibition of SYK kinase.

Comparison of effects of midostaurin, crenolanib, quizartinib, gilteritinib, sorafenib and BLU-285 on oncogenic mutants of KIT, CBL and FLT3 in haematological malignancies.

Mutations in two type-3 receptor tyrosine kinases (RTKs), KIT and FLT3, are common in both acute myeloid leukaemia (AML) and systemic mastocytosis (SM) and lead to hyperactivation of key signalling pathways. A large number of tyrosine kinase inhibitors (TKIs) have been developed that target either FLT3 or KIT and significant clinical benefit has been demonstrated in multiple clinical trials. Given the structural similarity of FLT3 and KIT, it is not surprising that some of these TKIs inhibit both of these receptors. This is typified by midostaurin, which has been approved by the US Food and Drug Administration for mutant FLT3-positive AML and for KIT D816V-positive SM. Here, we compare the in vitro activities of the clinically available FLT3 and KIT inhibitors with those of midostaurin against a panel of cells expressing a variety of oncogenic FLT3 or KIT receptors, including wild-type (wt) FLT3, FLT3-internal tandem duplication (ITD), FLT3 D835Y, the resistance mutant FLT3-ITD+ F691L, KIT D816V, and KIT N822K. We also examined the effects of these inhibitors in vitro and in vivo on cells expressing mutations in c-CBL found in AML that result in hypersensitization of RTKs, such as FLT3 and KIT. The results show a wide spectrum of activity of these various mutations to these clinically available TKIs.

Expression of the eight GABAA receptor α subunits in the developing zebrafish central nervous system.

GABA is a robust regulator of both developing and mature neural networks. It exerts many of its effects through GABAA receptors, which are heteropentamers assembled from a large array of subunits encoded by distinct genes. In mammals, there are 19 different GABAA subunit types, which are divided into the α, ß, γ, δ, ε, π, θ and ρ subfamilies. The immense diversity of GABAA receptors is not fully understood. However, it is known that specific isoforms, with their distinct biophysical properties and expression profiles, tune responses to GABA. Although larval zebrafish are well-established as a model system for neural circuit analysis, little is known about GABAA receptors diversity and expression in this system. Here, using database analysis, we show that the zebrafish genome contains at least 23 subunits. All but the mammalian θ and ε subunits have at least one zebrafish ortholog, while five mammalian GABAA receptor subunits have two zebrafish orthologs. Zebrafish contain one subunit, ß4, which does not have a clear mammalian ortholog. Similar to mammalian GABAA receptors, the zebrafish α subfamily is the largest and most diverse of the subfamilies. In zebrafish there are eight α subunits, and RNA in situ hybridization across early zebrafish development revealed that they demonstrate distinct patterns of expression in the brain, spinal cord, and retina. Some subunits were very broadly distributed, whereas others were restricted to small populations of cells. Subunit-specific expression patterns in zebrafish resembled were those found in frogs and rodents, which suggests that the roles of different GABAA receptor isoforms are largely conserved among vertebrates. This study provides a platform to examine isoform specific roles of GABAA receptors within zebrafish neural circuits and it highlights the potential of this system to better understand the remarkable heterogeneity of GABAA receptors.