The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.translational research | PCI-32765 | Imbruvica

In the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), NF-κB activity is essential for viability of the malignant cells and is sustained by constitutive activity of IκB kinase (IKK) in the cytoplasm. Here, we report an unexpected role for the bromodomain and extraterminal domain (BET) proteins BRD2 and BRD4 in maintaining oncogenic IKK activity in ABC DLBCL. IKK activity was reduced by small molecules targeting BET proteins as well as by genetic knockdown of BRD2 and BRD4 expression, thereby inhibiting downstream NF-κB-driven transcriptional programs and killing ABC DLBCL cells. Using a high-throughput platform to screen for drug-drug synergy, we observed that the BET inhibitor JQ1 combined favorably with multiple drugs targeting B-cell receptor signaling, one pathway that activates IKK in ABC DLBCL. The BTK kinase inhibitor ibrutinib, which is in clinical development for the treatment of ABC DLBCL, synergized strongly with BET inhibitors in killing ABC DLBCL cells in vitro and in a xenograft mouse model. These findings provide a mechanistic basis for the clinical development of BET protein inhibitors in ABC DLBCL, particularly in combination with other modulators of oncogenic IKK signaling.cancer therapy | drug synergism T he activated B-cell like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL) has an aggressive clinical course compared with other DLBCL subtypes, with an overall survival of only 40% with current multidrug chemotherapies (1, 2). In recent years, detailed genetic and functional genomic analyses unveiled the key oncogenic mechanisms that sustain the aggressiveness of this subtype. Notably, all ABC DLBCLs rely on constitutive NF-κB activation for survival (3). Various oncogenic events converge on NF-κB to promote lymphomagenesis. About 10% of ABC DLBCL tumors have activating mutations affecting CARD11, a scaffolding protein required for the assembly of the CARD11-BCL10-MALT1 (CBM) complex. Mutant CARD11 proteins spontaneously generate cytoplasmic CBM aggregates that drive constitutive NF-κB activity (4). ABC DLBCL tumors with wild-type CARD11 use other mechanisms to activate NF-κB. In 20% of cases, signals emanating from the B-cell receptor (BCR) are augmented by somatically acquired mutations targeting the BCR subunits CD79A and CD79B (5). In 39% of ABC DLBCLs, NF-κB is activated by somatic mutations targeting MyD88, an adaptor protein in the Toll-like receptor (TLR) pathway (6). In normal B cells, stimulus-dependent engagement of the BCR and MyD88 pathways activates IκB kinase (IKK), which phosphorylates ΙκBα, thereby promoting its degradation and allowing NF-κB transcription factors to enter the nucleus and activate a distinctive set of target genes. By contrast, ABC DLBCL cells become addicted to constitutive activity of IKK such that its inhibition is lethal (7). Recent therapeutic efforts to target oncogenic signaling in ABC DLBCL have focused on ibrutinib, a selective inhibitor of the kinase BTK that transmits signals from the BCR to the NF-κB pathway (5)...

Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. HTLV-1-encoded protein Tax (transactivator from the X-gene region) up-regulates Bcl-xL (B-cell lymphoma-extra large) expression and activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems, resulting in amplified JAK/STAT signaling. Inhibition of JAK signaling reduces cytokinedependent ex vivo proliferation of peripheral blood mononuclear cells (PBMCs) from ATL patients in smoldering/chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in ATL cell lines. The selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen combined with >450 potential therapeutic agents, and Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. The combination was noted to strongly activate BAX (Bcl-2-associated X protein), effect mitochondrial depolarization, and increase caspase 3/7 activities that lead to cleavage of PARP (poly ADP ribose polymerase) and Mcl-1 (myeloid cell leukemia 1). Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy, whereas the combination dramatically lowered tumor burden and prolonged survival in an ATL murine model. This combination strongly blocked ex vivo proliferation of five ATL patients' PBMCs. These studies provide support for a therapeutic trial in patients with smoldering/chronic ATL using a drug combination that inhibits JAK signaling and antiapoptotic protein Bcl-xL.adult T-cell leukemia | Janus kinase | ruxolitinib | navitoclax T -cell leukemia/lymphoma represents ∼10% of lymphoid malignancies. Genetic alterations affecting members of the Janus kinases (JAKs) and signal transducers and activators of transcription (STAT) were discovered in a variety of T-cell malignancies (1, 2). Furthermore, increases in the common γc cytokine concentrations that signal through the JAK1/3, STAT3/5 pathway have been demonstrated in angioimmunoblastic T-cell lymphoma, gamma delta T-cell lymphoma, and adult T-cell leukemia (ATL), thereby identifying effective therapeutic targets.ATL is an aggressive T-cell malignancy characterized by the clonal expansion of CD4 + CD25 + T lymphocytes that develops in a small proportion of individuals infected with human T-cell lymphotrophic virus-1 (HTLV-1) (3-5). Clinically, ATL is subclassified into four subtypes: smoldering, chronic, lymphomatous, and acute (4, 5). Presently, there is no curative therapy for ATL (4, 5). In search of effective therapies, we examined key signaling pathways that confer a proliferation and viability advantage to ATL cells. It was noted that the HTLV-1-encoded Tax (transactivator from the X-gene region) is associated with increased Bcl-xL (B-cell lymphoma-extra large) expression in ATL cells (6). Furthermore, we reported two autocrine loops (IL-2/IL-2Rα, IL-15/IL-15Rα) and one paracrine loop that in...

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials.

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

The RAS/RAF/MEK/ERK pathway is activated in greater than 30% of human cancers primarily via mutations in KRAS, NRAS and BRAF. The development of highly selective and genetic context-specific RAF inhibitors has led to spectacular improvements in patients with BRAFV600-mutant disease. Unfortunately, the BRAF V600E selectivity of these inhibitors prevents their utility in RAS-mutant tumors. Here, we describe an unexpected paralog-selectivity for the type II RAF inhibitor LXH254 that is currently undergoing clinical trials in RAS-driven tumors in combination with MEK and ERK inhibitors. LXH254 is a potent inhibitor of both monomeric and dimeric BRAF and CRAF, but a comparably poor inhibitor of ARAF.This paralog selectivity permits robust inhibition of dimeric BRAF and CRAF simultaneous with potentially improved TI.This tolerability profile has enabled vertical combinations with MEK and ERK inhibitors for the treatment of RAS-driven tumors in clinic.Research.

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAF alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors. In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling..

Direct pharmacological inhibition of RAS has remained elusive, and efforts to target CRAF have been challenging due to the complex nature of RAF signaling, downstream of activated RAS, and the poor overall kinase selectivity of putative RAF inhibitors. Herein, we describe 15 (LXH254, Aversa, R.; et al. Int. Patent WO2014151616A1, 2014), a selective continued...