Systemic Loss of C-terminal Src Kinase Expression Elicits Spontaneous Suppurative Inflammation in Conditional Knockout Mice.

C-terminal Src kinase (Csk) is one of the critical negative regulators of the Src family of kinases. The Src family of kinases are nonreceptor tyrosine kinases that regulate inflammation, cell proliferation, motility, and adhesion. To investigate potential histologic lesions associated with systemic loss of Csk gene activity in adult mice, conditional Csk-knockout mice were examined. Cre-mediated systemic excision of Csk induced by tamoxifen treatment resulted in multiorgan inflammation. Specifically, induction of Csk gene excision with three days of tamoxifen treatment resulted in greater than 90% gene excision. Strikingly, these mice developed enteritis that ranged from minimal and suppurative to severe, fibrinonecrosuppurative and hemorrhagic. Other inflammatory lesions included suppurative pneumonia, gastritis, and myocarditis, and increased numbers of inflammatory cells within the hepatic parenchyma. When tamoxifen treatment was reduced from three days to one day in an effort to lower the level of Csk gene excision and limit lesion development, the mice developed severe suppurative to pyogranulomatous pneumonia and minimal to mild suppurative enteritis. Lesions observed secondary to Csk gene excision suggest important roles for Csk in downregulating the proinflammatory activity of the Src family of kinases and limiting neutrophil-mediated inflammation.

F-box-directed CRL complex assembly and regulation by the CSN and CAND1.

The COP9 signalosome (CSN) is thought to maintain the stability of cullin-RING ubiquitin ligases (CRL) by limiting the autocatalytic destruction of substrate adapters such as F box proteins (FBPs). CAND1, a protein associated with unneddylated CUL1, was proposed to assist in this role in an as yet unclear fashion. We found that only a subset of Schizosaccharomyces pombe FBPs, which feature a critical F box proline that promotes their interaction with CUL1, required CSN for stability. Unlike the CRL3 adaptor Btb3p, none of the CSN-sensitive FBPs were affected by deletion of ubp12. Contrary to current models, CAND1 does not control adaptor stability but maintains the cellular balance of CRL1 complexes by preventing rare FBPs from being outcompeted for binding to CUL1 by more ample adapters. These findings were integrated into a refined model of CRL control in which substrate availability toggles CRLs between independent CSN and CAND1 cycles.

Discovery of (4-Pyrazolyl)-2-aminopyrimidines as Potent and Selective Inhibitors of Cyclin-Dependent Kinase 2.

CDK2 is a critical regulator of the cell cycle. For a variety of human cancers, the dysregulation of CDK2/cyclin E1 can lead to tumor growth and proliferation. Historically, early efforts to develop CDK2 inhibitors with clinical applications proved unsuccessful due to challenges in achieving selectivity over off-target CDK isoforms with associated toxicity. In this report, we describe the discovery of (4-pyrazolyl)-2-aminopyrimidines as a potent class of CDK2 inhibitors that display selectivity over CDKs 1, 4, 6, 7, and 9. SAR studies led to the identification of compound 17, a kinase selective and highly potent CDK2 inhibitor (IC50 = 0.29 nM). The evaluation of 17 in CCNE1-amplified mouse models shows the pharmacodynamic inhibition of CDK2, measured by reduced Rb phosphorylation, and antitumor activity.

Discovery of Potent, Dual-Inhibitors of Diacylglycerol Kinases Alpha and Zeta Guided by Phenotypic Optimization.

We describe a phenotypic screening and optimization strategy to discover compounds that block intracellular checkpoint signaling in T-cells. We identified dual DGKα and ζ inhibitors notwithstanding the modest similarity between α and ζ relative to other DGK isoforms. Optimized compounds produced cytokine release and T-cell proliferation consistent with DGK inhibition and potentiated an immune response in human and mouse T-cells. Additionally, lead inhibitor BMS-502 demonstrated dose-dependent immune stimulation in the mouse OT-1 model, setting the stage for a drug discovery program.

DGKα/ζ inhibitors combine with PD-1 checkpoint therapy to promote T cell-mediated antitumor immunity.

Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses. Toward this end, we designed a human primary T cell phenotypic high-throughput screening strategy to identify small molecules with distinct and complementary mechanisms of action to PD-1 checkpoint blockade. Through these efforts, we selected and optimized a chemical series that showed robust potentiation of T cell activation and combinatorial activity with αPD-1 blockade. Target identification was facilitated by chemical proteomic profiling with a lipid-based photoaffinity probe, which displayed enhanced binding to diacylglycerol kinase α (DGKα) in the presence of the active compound, a phenomenon that correlated with the translocation of DGKα to the plasma membrane. We further found that optimized leads within this chemical series were potent and selective inhibitors of both DGKα and DGKζ, lipid kinases that constitute an intracellular T cell checkpoint that blunts T cell signaling through diacylglycerol metabolism. We show that dual DGKα/ζ inhibition amplified suboptimal T cell receptor signaling mediated by low-affinity antigen presentation and low major histocompatibility complex class I expression on tumor cells, both hallmarks of resistance to PD-1 blockade. In addition, DGKα/ζ inhibitors combined with αPD-1 therapy to elicit robust tumor regression in syngeneic mouse tumor models. Together, these findings support targeting DGKα/ζ as a next-generation T cell immune checkpoint strategy.

CSN facilitates Cullin-RING ubiquitin ligase function by counteracting autocatalytic adapter instability.

The COP9 signalosome (CSN) is known to bind cullin-RING ubiquitin ligases (CRLs) and to promote their activity in vivo. The mechanism of this stimulation has remained enigmatic because CSN's intrinsic and associated enzymatic activities paradoxically inhibit CRL activity in vitro. Reconciling this paradox, we show here that Csn5-catalysed cullin (Cul) deneddylation and Ubp12-mediated deubiquitination cooperate in maintaining the stability of labile substrate adapters, thus facilitating CRL function. Various fission-yeast csn and ubp12 deletion mutants have lower levels of the Cul3p adapter Btb3p. This decrease is due to increased autocatalytic, Cul3p-dependent, ubiquitination and the subsequent degradation of Btb3p. The CSN-Ubp12p pathway also maintains the stability of the Cul1p adapter Pop1p, a mechanism required for the efficient destruction of its cognate substrate Rum1p. Emphasizing the physiological importance of this mechanism, we found that the dispensable csn5 and ubp12 genes become essential for viability when adapter recruitment to Cul1p is compromised. Our data suggest that maintenance of adapter stability is a general mechanism of CRL control by the CSN.

Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells.

NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235.

The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases?

The COP9 signalosome (CSN) is a highly conserved protein complex implicated in diverse biological functions that involve ubiquitin-mediated proteolysis. Paradoxically, conserved enzymatic activities associated with CSN inhibit cullin ubiquitin ligase activity in vitro, whereas mutational analysis suggests that CSN promotes cullin-dependent proteolysis in vivo. This apparent paradox can be resolved in a model that proposes CSN-mediated cullin inhibition is a prerequisite for the proper assembly and maintenance of active cullin ubiquitin ligase complexes.

PTEN-deficient cancers depend on PIK3CB.

Deregulation of the PI3K signaling pathway is observed in many human cancers and occurs most frequently through loss of PTEN phosphatase tumor suppressor function or through somatic activating mutations in the Class IA PI3K, PIK3CA. Tumors harboring activated p110alpha, the protein product of PIK3CA, require p110alpha activity for growth and survival and hence are expected to be responsive to inhibitors of its lipid kinase activity. Whether PTEN-deficient cancers similarly depend on p110alpha activity to sustain activation of the PI3K pathway has been unclear. In this study, we used a single-vector lentiviral inducible shRNA system to selectively inactivate the three Class IA PI3Ks, PIK3CA, PIK3CB, and PIK3CD, to determine which PI3K isoforms are responsible for driving the abnormal proliferation of PTEN-deficient cancers. Down-regulation of PIK3CA in colorectal cancer cells harboring mutations in PIK3CA inhibited downstream PI3K signaling and cell growth. Surprisingly, PIK3CA depletion affected neither PI3K signaling nor cell growth in 3 PTEN-deficient cancer cell lines. In contrast, down-regulation of the PIK3CB isoform, which encodes p110beta, resulted in pathway inactivation and subsequent inhibition of growth in both cell-based and in vivo settings. This essential function of PIK3CB in PTEN-deficient cancer cells required its lipid kinase activity. Our findings demonstrate that although p110alpha activation is required to sustain the proliferation of established PIK3CA-mutant tumors, PTEN-deficient tumors are dependent instead on p110beta signaling. This unexpected finding demonstrates the need to tailor therapeutic approaches to the genetic basis of PI3K pathway activation to achieve optimal treatment response.

DGKζ exerts greater control than DGKα over CD8+ T cell activity and tumor inhibition.

Two isoforms of diacylglycerol kinases (DGKs), DGKα and DGKζ, are primarily responsible for terminating DAG-mediated activation of Ras and PKCθ pathways in T cells. A direct comparison of tumor growth between mice lacking each isoform has not been undertaken. We evaluated the growth of three syngeneic tumor cell lines in mice lacking either DGKα or DGKζ in the presence or absence of treatment with anti-PD1 and determined that (i) mice deficient in DGKζ conferred enhanced control of tumor relative to mice deficient in DGKα and (ii) deficiency of DGKζ acted additively with anti-PD1 in tumor control. Consistent with this finding, functional and RNA-sequencing analyses revealed greater changes in stimulated DGKζ-deficient T cells compared with DGKα-deficient T cells, which were enhanced relative to wildtype T cells. DGKζ also imparted greater regulation than DGKα in human T cells. Together, these data support targeting the ζ isoform of DGKs to therapeutically enhance T cell anti-tumor activity.

Discovery and Preclinical Pharmacology of an Oral Bromodomain and Extra-Terminal (BET) Inhibitor Using Scaffold-Hopping and Structure-Guided Drug Design.

Inhibition of the bromodomain and extra-terminal (BET) family of adaptor proteins is an attractive strategy for targeting transcriptional regulation of key oncogenes, such as c-MYC. Starting with the screening hit 1, a combination of structure-activity relationship and protein structure-guided drug design led to the discovery of a differently oriented carbazole 9 with favorable binding to the tryptophan, proline, and phenylalanine (WPF) shelf conserved in the BET family. Identification of an additional lipophilic pocket and functional group optimization to optimize pharmacokinetic (PK) properties culminated in the discovery of 18 (BMS-986158) with excellent potency in binding and functional assays. On the basis of its favorable PK profile and robust in vivo activity in a panel of hematologic and solid tumor models, BMS-986158 was selected as a candidate for clinical evaluation.

Why is cancer drug discovery so difficult?

Thirty-five years after the 'war on cancer' was declared, the discovery of anticancer drugs remains a highly challenging endeavour. Here, we consider the factors responsible, such as tumour heterogeneity, and suggest strategies to improve the chances of short-term success in the development of novel anticancer drugs.

Discovery of Pyridazinone and Pyrazolo[1,5-a]pyridine Inhibitors of C-Terminal Src Kinase.

C-terminal Src kinase (CSK) functions as a negative regulator of T cell activation through inhibitory phosphorylation of LCK, so inhibitors of CSK are of interest as potential immuno-oncology agents. Screening of an internal kinase inhibitor collection identified pyridazinone lead 1, and a series of modifications led to optimized compound 13. Compound 13 showed potent activity in biochemical and cellular assays in vitro and demonstrated the ability to increase T cell proliferation induced by T cell receptor signaling. Compound 13 gave extended exposure in mice upon oral dosing and produced a functional response (decrease in LCK phosphorylation) in mouse spleens at 6 h post dose.

Inhibition of Casein Kinase 2 Disrupts Differentiation of Myeloid Cells in Cancer and Enhances the Efficacy of Immunotherapy in Mice.

The role of myeloid cells as regulators of tumor progression that significantly impact the efficacy of cancer immunotherapies makes them an attractive target for inhibition. Here we explore the effect of a novel, potent, and selective inhibitor of serine/threonine protein kinase casein kinase 2 (CK2) on modulating myeloid cells in the tumor microenvironment. Although inhibition of CK2 caused only a modest effect on dendritic cells in tumor-bearing mice, it substantially reduced the amount of polymorphonuclear myeloid-derived suppressor cells and tumor-associated macrophages. This effect was not caused by the induction of apoptosis, but rather by a block of differentiation. Our results implicated downregulation of CCAAT-enhancer binding protein-α in this effect. Although CK2 inhibition did not directly affect tumor cells, it dramatically enhanced the antitumor activity of immune checkpoint receptor blockade using anti-CTLA-4 antibody. These results suggest a potential role of CK2 inhibitors in combination therapies against cancer.Significance: These findings demonstrate the modulatory effects of casein kinase 2 inhibitors on myeloid cell differentiation in the tumor microenvironment, which subsequently synergize with the antitumor effects of checkpoint inhibitor CTLA4. Cancer Res; 78(19); 5644-55. ©2018 AACR.

PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes.

BACKGROUND: PCI/MPN domain protein complexes comprise the 19S proteasome lid, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex is thought to be composed of essential core subunits required for global protein synthesis and non-essential subunits that may modulate mRNA specificity. Interactions of unclear significance were reported between eIF3 subunits and PCI proteins contained in the CSN. RESULTS: Here, we report the unexpected finding that fission yeast has two distinct eIF3 complexes sharing common core subunits, but distinguished by the PCI proteins eIF3e and the novel eIF3m, which was previously annotated as a putative CSN subunit. Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation. Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes. Whereas the eIF3m complex appears to associate with the bulk of cellular mRNAs, the eIF3e complex associates with a far more restricted set. The microarray findings were independently corroborated for a random set of 14 mRNAs by RT-PCR analysis. CONCLUSION: We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs.

Sensitivity of Small Cell Lung Cancer to BET Inhibition Is Mediated by Regulation of ASCL1 Gene Expression.

The BET (bromodomain and extra-terminal) proteins bind acetylated histones and recruit protein complexes to promote transcription elongation. In hematologic cancers, BET proteins have been shown to regulate expression of MYC and other genes that are important to disease pathology. Pharmacologic inhibition of BET protein binding has been shown to inhibit tumor growth in MYC-dependent cancers, such as multiple myeloma. In this study, we demonstrate that small cell lung cancer (SCLC) cells are exquisitely sensitive to growth inhibition by the BET inhibitor JQ1. JQ1 treatment has no impact on MYC protein expression, but results in downregulation of the lineage-specific transcription factor ASCL1. SCLC cells that are sensitive to JQ1 are also sensitive to ASCL1 depletion by RNAi. Chromatin immunoprecipitation studies confirmed the binding of the BET protein BRD4 to the ASCL1 enhancer, and the ability of JQ1 to disrupt the interaction. The importance of ASCL1 as a potential driver oncogene in SCLC is further underscored by the observation that ASCL1 is overexpressed in >50% of SCLC specimens, an extent greater than that observed for other putative oncogenes (MYC, MYCN, and SOX2) previously implicated in SCLC. Our studies have provided a mechanistic basis for the sensitivity of SCLC to BET inhibition and a rationale for the clinical development of BET inhibitors in this disease with high unmet medical need.

Conservation of the COP9/signalosome in budding yeast.

BACKGROUND: The COP9/signalosome (CSN), a multiprotein complex consisting of eight subunits, is implicated in a wide variety of regulatory processes including cell cycle control, signal transduction, transcriptional activation, and plant photomorphogenesis. Some of these functions have been linked to CSN-associated enzymes, including kinases and an activity that removes the ubiquitin-like protein NEDD8/Rub1p from the cullin subunit of E3 ligases. CSN is highly conserved across species from fission yeast to humans, but sequence comparison has failed to identify the complex in budding yeast, except for a putative CSN5 subunit called Rri1p. RESULTS: We show that disruption of four budding yeast genes, PCI8 and three previously uncharacterized ORFs, which encode proteins interacting with Rrr1p/Csn5p, each results in the accumulation of the cullin Cdc53p exclusively in the Rub1p-modified state. This phenotype, which resembles that of fission yeast csn mutants, is due to a biochemical defect in deneddylation that is complemented by wild-type cell lysate and by purified human CSN in vitro. Although three of the four genes encode proteins with PCI domains conserved in metazoan CSN proteins, their disruption does not confer the DNA damage sensitivity described in some fission yeast csn mutants. CONCLUSIONS: Our studies present unexpected evidence for the conservation of a functional homologue of the metazoan CSN, which mediates control of cullin neddylation in budding yeast.

Targeting epigenetic regulators for cancer therapy.

Human gene expression patterns are controlled and coordinated by the activity of a diverse array of epigenetic regulators, including histone methyltransferases, acetyltransferases, and chromatin remodelers. Deregulation of these epigenetic pathways can lead to genome-wide changes in gene expression, with serious disease consequences. In recent years, research has suggested that cross talk between genomic (i.e., for example, mutations, translocations) and epigenomic factors may drive the etiology of both hematologic malignancies and solid tumors. Current work in translational research seeks to identify epigenetic regulators whose aberrant activity contributes to oncogenesis, including the histone methyltransferases DOT1L and EZH2 and the bromodomain-containing BET family, and to develop drugs that inhibit the aberrant activity of these regulators. Preclinical and clinical studies using small-molecule inhibitors of epigenetic regulators have underscored their value for therapeutic intervention, and these inhibitors can also be used to drive further studies into dissecting the functions of epigenetic factors in normal and cancer cells.

PI3K pathway activation mediates resistance to MEK inhibitors in KRAS mutant cancers.

The RAS pathway is one of the most frequently deregulated pathways in cancer. RAS signals through multiple effector pathways, including the RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK and phosphatidylinositol 3-kinase (PI3K)-AKT signaling cascades. The oncogenic potential of these effector pathways is illustrated by the frequent occurrence of activating mutations in BRAF and PIK3CA as well as loss-of-function mutations in the tumor suppressor PTEN, a negative regulator of PI3K. Previous studies have found that whereas BRAF mutant cancers are highly sensitive to MEK inhibition, RAS mutant cancers exhibit a more variable response. The molecular mechanisms responsible for this heterogeneous response remain unclear. In this study, we show that PI3K pathway activation strongly influences the sensitivity of RAS mutant cells to MEK inhibitors. Activating mutations in PIK3CA reduce the sensitivity to MEK inhibition, whereas PTEN mutations seem to cause complete resistance. We further show that down-regulation of PIK3CA resensitizes cells with co-occurring KRAS and PIK3CA mutations to MEK inhibition. At the molecular level, the dual inhibition of both pathways seems to be required for complete inhibition of the downstream mammalian target of rapamycin effector pathway and results in the induction of cell death. Finally, we show that whereas inactivation of either the MEK or PI3K pathway leads to partial tumor growth inhibition, targeted inhibition of both pathways is required to achieve tumor stasis. Our study provides molecular insights that help explain the heterogeneous response of KRAS mutant cancers to MEK pathway inhibition and presents a strong rationale for the clinical testing of combination MEK and PI3K targeted therapies.

Single-vector inducible lentiviral RNAi system for oncology target validation.

The use of RNA interference (RNAi) has enabled loss-of-function studies in mammalian cancer cells and has hence become critical for identifying and validating cancer drug targets. Current transient siRNA and stable shRNA systems, however, have limited utility in accurately assessing the cancer dependency due to their short-lived effects and limited in vivo utility, respectively. In this study, a single-vector lentiviral, Tet-inducible shRNA system (pLKO-Tet-On) was generated to allow for the rapid generation of multiple stable cell lines with regulatable shRNA expression. We demonstrate the advantages and versatility of this system by targeting two polycomb group proteins, Bmi-1 and Mel-18, in a number of cancer cell lines. Our data show that pLKO-Tet-On-mediated knockdown is tightly regulated by the inducer tetracycline and its derivative, doxycycline, in a concentration- and time-dependent manner. Furthermore, target gene expression is fully restored upon withdrawal of the inducing agent. An additional, 17 distinct gene products have been targeted by inducible shRNAs with robust regulation in all cases. Importantly, we functionally validate the ability of the pLKO-Tet-On vector to reversibly silence targeted transcripts in vivo. The versatile and robust inducible lentiviral RNAi system reported herein can therefore serve as a powerful tool to rapidly reveal tumor cell dependence.