Discovery of an Allosteric, Inactive Conformation-Selective Inhibitor of Full-Length HPK1 Utilizing a Kinase Cascade Assay.

Achieving selectivity across the human kinome is a major hurdle in kinase inhibitor drug discovery. Assays using active, phosphorylated protein kinases bias hits toward poorly selective inhibitors that bind within the highly conserved adenosine triphosphate (ATP) pocket. Targeting inactive (vs active) kinase conformations offers advantages in achieving selectivity because of their more diversified structures. Kinase cascade assays are typically initiated with target kinases in their unphosphorylated inactive forms, which are activated during the assays. Therefore, these assays are capable of identifying inhibitors that preferentially bind to the unphosphorylated form of the enzyme in addition to those that bind to the active form. We applied this cascade assay to the emerging cancer immunotherapy target hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase that negatively regulates T cell receptor signaling. Using this approach, we discovered an allosteric, inactive conformation-selective triazolopyrimidinone HPK1 inhibitor, compound 1. Compound 1 binds to unphosphorylated HPK1 >24-fold more potently than active HPK1, is not competitive with ATP, and is highly selective against kinases critical for T cell signaling. Furthermore, compound 1 does not bind to the isolated HPK1 kinase domain alone but requires other domains. Together, these data indicate that 1 is an allosteric HPK1 inhibitor that attenuates kinase autophosphorylation by binding to a pocket consisting of residues within and outside of the kinase domain. Our study demonstrates that cascade assays can lead to the discovery of highly selective kinase inhibitors. The triazolopyrimidinone described in this study may represent a privileged chemical scaffold for further development of potent and selective HPK1 inhibitors.

Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery.

In both academia and the pharmaceutical industry, large-scale assays for drug discovery are expensive and often impractical, particularly for the increasingly important physiologically relevant model systems that require primary cells, organoids, whole organisms, or expensive or rare reagents. We hypothesized that data from a single high-throughput imaging assay can be repurposed to predict the biological activity of compounds in other assays, even those targeting alternate pathways or biological processes. Indeed, quantitative information extracted from a three-channel microscopy-based screen for glucocorticoid receptor translocation was able to predict assay-specific biological activity in two ongoing drug discovery projects. In these projects, repurposing increased hit rates by 50- to 250-fold over that of the initial project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from high-content screens are a rich source of information that can be used to predict and replace customized biological assays.

Discovery and Pharmacological Characterization of JNJ-42756493 (Erdafitinib), a Functionally Selective Small-Molecule FGFR Family Inhibitor.

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010-20. ©2017 AACR.

Tankyrase inhibition impairs directional migration and invasion of lung cancer cells by affecting microtubule dynamics and polarity signals.

BACKGROUND: Tankyrases are poly(adenosine diphosphate)-ribose polymerases that contribute to biological processes as diverse as modulation of Wnt signaling, telomere maintenance, vesicle trafficking, and microtubule-dependent spindle pole assembly during mitosis. At interphase, polarized reshaping of the microtubule network fosters oriented cell migration. This is attained by association of adenomatous polyposis coli with the plus end of microtubules at the cortex of cell membrane protrusions and microtubule-based centrosome reorientation towards the migrating front. RESULTS: Here we report a new function for tankyrases, namely, regulation of directional cell locomotion. Using a panel of lung cancer cell lines as a model system, we found that abrogation of tankyrase activity by two different, structurally unrelated small-molecule inhibitors (one introduced and characterized here for the first time) or by RNA interference-based genetic silencing weakened cell migration, invasion, and directional movement induced by the motogenic cytokine hepatocyte growth factor. Mechanistically, the anti-invasive outcome of tankyrase inhibition could be ascribed to sequential deterioration of the distinct events that govern cell directional sensing. In particular, tankyrase blockade negatively impacted (1) microtubule dynamic instability; (2) adenomatous polyposis coli plasma membrane targeting; and (3) centrosome reorientation. CONCLUSIONS: Collectively, these findings uncover an unanticipated role for tankyrases in influencing at multiple levels the interphase dynamics of the microtubule network and the subcellular distribution of related polarity signals. These results encourage the further exploration of tankyrase inhibitors as therapeutic tools to oppose dissemination and metastasis of cancer cells.

Convergent mutations and kinase fusions lead to oncogenic STAT3 activation in anaplastic large cell lymphoma.

A systematic characterization of the genetic alterations driving ALCLs has not been performed. By integrating massive sequencing strategies, we provide a comprehensive characterization of driver genetic alterations (somatic point mutations, copy number alterations, and gene fusions) in ALK(-) ALCLs. We identified activating mutations of JAK1 and/or STAT3 genes in ∼20% of 88 [corrected] ALK(-) ALCLs and demonstrated that 38% of systemic ALK(-) ALCLs displayed double lesions. Recurrent chimeras combining a transcription factor (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were also discovered in WT JAK1/STAT3 ALK(-) ALCL. All these aberrations lead to the constitutive activation of the JAK/STAT3 pathway, which was proved oncogenic. Consistently, JAK/STAT3 pathway inhibition impaired cell growth in vitro and in vivo.

Upregulation of MAPK Negative Feedback Regulators and RET in Mutant ALK Neuroblastoma: Implications for Targeted Treatment.

PURPOSE: Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies. EXPERIMENTAL DESIGN: To achieve this goal, transcriptome profiling was performed in neuroblastoma cell lines with the ALK(F1174L) or ALK(R1275Q) hotspot mutations, ALK amplification, or wild-type ALK following pharmacologic inhibition of ALK using four different compounds. Next, we performed cross-species genomic analyses to identify commonly transcriptionally perturbed genes in MYCN/ALK(F1174L) double transgenic versus MYCN transgenic mouse tumors as compared with the mutant ALK-driven transcriptome in human neuroblastomas. RESULTS: A 77-gene ALK signature was established and successfully validated in primary neuroblastoma samples, in a neuroblastoma cell line with ALK(F1174L) and ALK(R1275Q) regulable overexpression constructs and in other ALKomas. In addition to the previously established PI3K/AKT/mTOR, MAPK/ERK, and MYC/MYCN signaling branches, we identified that mutant ALK drives a strong upregulation of MAPK negative feedback regulators and upregulates RET and RET-driven sympathetic neuronal markers of the cholinergic lineage. CONCLUSIONS: We provide important novel insights into the transcriptional consequences and the complexity of mutant ALK signaling in this aggressive pediatric tumor. The negative feedback loop of MAPK pathway inhibitors may affect novel ALK inhibition therapies, whereas mutant ALK induced RET signaling can offer novel opportunities for testing ALK-RET oriented molecular combination therapies.

Activation of RAS family members confers resistance to ROS1 targeting drugs.

The ROS1 tyrosine kinase is activated in lung cancer as a consequence of chromosomal rearrangement. Although high response rates and disease control have been observed in lung cancer patients bearing rearranged ROS1 tumors (ROS1+) treated with the kinase inhibitor crizotinib, many of these patients eventually relapse.To identify mechanisms of resistance to ROS1 inhibitors we generated resistant cells from HCC78 lung cancer cells bearing the SLC34A2-ROS1 rearrangement. We found that activation of the RAS pathway in the HCC78 cell model, due to either KRAS/NRAS mutations or to KRAS amplification, rendered the cells resistant to ROS1 inhibition. These cells were cross-resistant to different ROS1 inhibitors, but sensitive to inhibitors of the RAS signaling pathway. Interestingly, we identified focal KRAS amplification in a biopsy of a tumor from a patient that had become resistant to crizotinib treatment.Altogether our data suggest that the activation of members of the RAS family can confer resistance to ROS1 inhibitors. This has important clinical implications as: (i) RAS genetic alterations in ROS1+ primary tumors are likely negative predictors of efficacy for targeted drugs and (ii) this kind of resistance is unlikely to be overcome by the use of more specific or more potent ROS1 targeting drugs.

p53-independent regulation of p21Waf1/Cip1 expression and senescence by Chk2.

The Chk2 kinase is a tumor suppressor and key component of the DNA damage checkpoint response that encompasses cell cycle arrest, apoptosis, and DNA repair. It has also been shown to have a role in replicative senescence resulting from dysfunctional telomeres. Some of these functions are at least partially exerted through activation of the p53 transcription factor. High-level expression of virally transduced Chk2 in A549 human lung carcinoma cells led to arrested proliferation, apoptosis, and senescence. These were accompanied by various molecular events, including p21(Waf1/Cip1) (p21) transcriptional induction, consistent with p53 activation. However, Chk2-dependent senescence and p21 transcriptional induction also occurred in p53-defective SK-BR-3 (breast carcinoma) and HaCaT (immortalized keratinocyte) cells. Small interfering RNA-mediated knockdown of p21 in p53-defective cells expressing Chk2 resulted in a decrease in senescent cells. These results revealed a p53-independent role for Chk2 in p21 induction and senescence that may contribute to tumor suppression and genotoxic treatment outcome.

Critical role of the ubiquitin ligase activity of UHRF1, a nuclear RING finger protein, in tumor cell growth.

Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or alteration in growth signaling pathways. Identification of novel genes involved in cellular proliferation may lead to new classes of cancer therapeutics. By screening a tetracycline-inducible cDNA library in A549 cells for genes that interfere with proliferation, we have identified a fragment of UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, that acts as a dominant negative effector of cell growth. Reduction of UHRF1 levels using an UHRF1-specific shRNA decreased growth rates in several tumor cell lines. In addition, treatment of A549 cells with agents that activated different cell cycle checkpoints resulted in down-regulation of UHRF1. The primary sequence of UHRF1 contains a PHD and a RING motif, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that UHRF1 displays RING-dependent E3 ligase activity. Overexpression of a GFP-fused UHRF1 RING mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics. Taken together, our results suggest a general requirement for UHRF1 in tumor cell proliferation and implicate the RING domain of UHRF1 as a functional determinant of growth regulation.

Disruption of murine Mus81 increases genomic instability and DNA damage sensitivity but does not promote tumorigenesis.

The Mus81-Eme1 endonuclease is implicated in the efficient rescue of broken replication forks in Saccharomyces cerevisiae and Schizosaccharomyces pombe. We have used gene targeting to study the function of the Mus81-Eme1 endonuclease in mammalian cells. Mus81-deficient mice develop normally and are fertile. Surprisingly, embryonic fibroblasts from Mus81(-/-) animals fail to proliferate in vitro. This proliferation defect can be rescued by expression of the papillomavirus E6 protein that promotes degradation of p53. When grown in culture, Mus81(-/-) cells have elevated levels of DNA damage, acquire chromosomal aberrations, and are hypersensitive to agents that generate DNA cross-links. In contrast to the situation in yeast, murine Mus81 is not required for replication restart following camptothecin treatment. Mus81(-/-) mice and cells are hypersensitive to DNA cross-linking agents. Cross-link-induced double-strand break formation is normal in Mus81(-/-) cells, but the resolution of repair intermediates is not. The persistence of Rad51 foci in Mus81(-/-) cells suggests that Mus81 acts at a late step in the repair of cross-link-induced lesions. Despite these defects, Mus81(-/-) mice do not show increased predisposition to lymphoma or any other malignancy in the first year of life.

Involvement of GSK-3beta in TWEAK-mediated NF-kappaB activation.

Glycogen synthase kinase-3beta (GSK-3beta) is a key component of several signaling pathways. We found that a short variant of 'TNF-like weak inducer of apoptosis' (shortTWEAK) formed a complex with GSK-3beta in a yeast two-hybrid system. We demonstrate that shortTWEAK and GSK-3beta colocalize in the nucleus of human neuroblastoma cells. We also show that TWEAK is internalized in different cell lines and that it translocates to the nucleus. This event causes the degradation of IkappaBalpha, the nuclear translocation of both GSK-3beta and p65, and the induction of NF-kappaB-driven gene expression. We demonstrate that the induction of IL-8 expression by TWEAK can be counteracted by LiCl. Taken together, these data suggest that GSK-3beta plays an important role in the signal transduction pathway between TWEAK and NF-kappaB.

The budding yeast Rad9 checkpoint complex: chaperone proteins are required for its function.

Rad9 functions in the DNA-damage checkpoint pathway of Saccharomyces cerevisiae. In whole-cell extracts, Rad9 is found in large, soluble complexes, which have functions in amplifying the checkpoint signal. The two main soluble forms of Rad9 complexes that are found in cells exposed to DNA-damaging treatments were purified to homogeneity. Both of these Rad9 complexes contain the Ssa1 and/or Ssa2 chaperone proteins, suggesting a function for these proteins in checkpoint regulation. Consistent with this possibility, genetic experiments indicate redundant functions for SSA1 and SSA2 in survival, G2/M-checkpoint regulation, and phosphorylation of both Rad9 and Rad53 after irradiation with ultraviolet light. Ssa1 and Ssa2 can now be considered as novel checkpoint proteins that are likely to be required for remodelling Rad9 complexes during checkpoint-pathway activation.