Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers of response to targeted agents. To uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines, which represent much of the tissue-type and genetic diversity of human cancers, with 130 drugs under clinical and preclinical investigation. In aggregate, we found mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing’s sarcoma cells harboring the EWS-FLI1 gene translocation to PARP inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.
Tumor oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state 1 , but direct pharmacological inhibition of transcription factors has thus far proven difficult 2 . However, the transcriptional machinery contains various enzymatic co-factors that can be targeted for development of new therapeutic candidates 3 , including cyclin-dependent kinases (CDKs) 4 . Here we present the discovery and characterization of the first covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell line profiling indicates that a subset of cancer cell lines, including T-ALL, exhibit exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and this transcription factor’s key role in the core transcriptional regulatory circuitry of these tumor cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumor types exhibiting extreme dependencies on transcription for maintenance of the oncogenic state.
ISG15 is an interferon (IFN)-␣/-induced ubiquitin-like protein that is conjugated to cellular proteins during innate immune responses to viral and bacterial infections.A recent proteomics study identified 158 human proteins targeted for ISG15 conjugation, including the ISG15 E1 and E2 enzymes (Ube1L and UbcH8, respectively) and a HECT E3 enzyme, Herc5. Like the genes encoding Ube1L and UbcH8, expression of Herc5 was also induced by IFN-, suggesting that Herc5 might be a component of the ISG15 conjugation system. Consistent with this, small interfering RNAs targeting Herc5 had a dramatic effect on overall ISG15 conjugation in human cells, abrogating conjugation to the vast majority of ISG15 target proteins in vivo. In addition, co-transfection of plasmids expressing ISG15, Ube1L, UbcH8, and Herc5 resulted in robust ISG15 conjugation in non-IFN-treated cells, while the active-site cysteine mutant of Herc5 or a mutant lacking the RCC1 repeat region did not support ISG15 conjugation. These results demonstrate that Herc5 is required for conjugation of ISG15 to a broad spectrum of target proteins in human cells.Type 1 interferons play an essential role in innate immunity (1). One of the many genes strongly activated by IFN 4 -␣/ encodes ISG15, a 15-kDa ubiquitin-like protein (Ubl) (2, 3). Like ubiquitin (Ub), Ubls are linked to target proteins via isopeptide bonds between their terminal carboxyl group and lysine side chains of target proteins (4). The fact that ISG15 is expressed and conjugated in IFN-␣/-stimulated cells and lipopolysaccharide-stimulated cells implies that ISG15 conjugation is likely to mediate an important component of the innate immune response. This is supported by the finding that the influenza B virus NS1B protein specifically blocks ISG15 conjugation (5).The biochemical effect of ISG15 on target proteins is unknown; however, the recent identification of a large number of target proteins (6) provides opportunities for determining both the function of ISG15 and its role in the innate immune response. Also essential for functional studies is the identification of the complete set of enzymes required for ISG15 conjugation. As with Ub conjugation, it is presumed that a cooperating set of E1, E2, and E3 enzymes, in addition to possible accessory factors, will be required for ISG15 conjugation. The ISG15 E1 and E2 enzymes have been identified. Ube1L is a single-subunit enzyme 62% similar to the Ub E1 enzyme (5), and UbcH8 is the major, if not exclusive, E2 enzyme for ISG15 (7,8). The genes encoding both Ube1L and UbcH8 are, like ISG15, transcriptionally activated by IFN-␣/ (5, 7-9), suggesting that the entire conjugation system might be coordinately regulated. A candidate E3 enzyme for ISG15 conjugation emerged from mass spectrometry-based identification of ISG15 target proteins (6). Proteomics analyses of SUMO-and Ub-conjugated proteins have shown that enzymatic components of Ub/Ubl conjugation pathways are often auto-conjugated (10, 11), and consistent with this, Ube1L and UbcH8 were identified ...
SummaryPersonalized cancer therapy is based on a patient’s tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient’s living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care.
Colorectal cancers (CRCs) harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that combines targeting of the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS mutant (MT) and BRAF MT but not wild-type (WT) CRC cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT CRCs, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS MT colorectal cancer xenograft and genetically-engineered mouse models of CRC, but not in the corresponding KRAS WT CRC models. These data suggest that the combination of BCL-2/XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.
SummaryFewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.
BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.small-cell lung cancer | targeted therapies | BH3 mimetics | apoptosis | BIM
The reaction cycle of HECT domain ubiquitin ligases consists of three steps: 1) binding of an E2 protein, 2) transfer of ubiquitin from E2 to the HECT domain, and 3) transfer of ubiquitin to the substrate. We report the identification of a determinant that is specifically required for the last step of this cycle, a phenylalanine residue located four amino acids from the C terminus of most HECT domains, referred to here as the ؊4F. Alteration of this residue in human E6AP and Saccharomyces cerevisae Rsp5p did not affect ubiquitin-thioester formation, but effectively blocked substrate ubiquitination. Alteration of the ؊4F to alanine with concomitant substitution of a nearby residue to phenylalanine only partially restored Rsp5p activity, indicating that precise spatial placement of this residue is important. C-terminally extended E6AP and Rsp5p proteins were also defective for substrate ubiquitination, providing a likely biochemical understanding of a previously isolated Angelman syndrome-associated mutation of E6AP that alters the stop codon of an otherwise wild-type gene. We propose that the ؊4F may play a role in orienting ubiquitin when it is tethered to the HECT active site cysteine. This may be necessary to allow for approach of the incoming lysine ⑀-amino group of the substrate.The best characterized function of protein ubiquitination is as a recognition signal for the 26 S proteasome (1). Ubiquitin is covalently linked to substrate proteins via isopeptide bond linkages formed between the terminal carboxyl group of ubiquitin and ⑀-amino groups of lysine side chains of the substrate, or in some cases to the N-terminal ␣-amino group. Additional ubiquitin molecules can be conjugated sequentially at specific lysine residues of ubiquitin to form multi-ubiquitinated proteins, which are the principal substrate of the 26 S proteasome.Three groups of proteins cooperate in catalyzing ubiquitination: the E1 ubiquitin-activating enzyme, the E2 ubiquitinconjugating proteins (Ubc 1 proteins), and the E3 ubiquitin ligases (1). The E1 enzyme uses ATP to form a ubiquitinadenylate intermediate at the terminal carboxyl group of ubiquitin. A ubiquitin-thioester is then formed at the active site cysteine of the E1 enzyme, with release of AMP. The E1 enzyme transfers ubiquitin to the active site cysteine of the highly conserved family of E2 proteins, maintaining a ubiquitin-thioester linkage. The E3 ubiquitin-ligases interact with both the E2 proteins as well as with substrate proteins. Whereas several classes of E3 enzymes are now recognized (SCF, APC, CBC, RING, CHIP, and HECT E3s), they fall into two groups with respect to overall mechanism. The first group, which includes all classes except for the HECT E3s, are thought to function essentially as docking proteins, orienting the E2 and substrate so that ⑀-amino groups of substrate lysines can attack the E2-ubiquitin thioester, forming a ubiquitin-isopeptide bond. HECT E3s participate more directly in catalysis and are defined by a conserved C-terminal domain of ϳ350 amino aci...
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