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Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4's core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4's substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds' cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4-expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability.

Phosphoinositide 3-kinase (PI3K) activity is stimulated by diverse oncogenes and growth factor receptors, and elevated PI3K signaling is considered a hallmark of cancer. Many PI3K pathway-targeted therapies have been tested in oncology trials, resulting in regulatory approval of one isoform-selective inhibitor (idelalisib) for treatment of certain blood cancers and a variety of other agents at different stages of development. In parallel to PI3K research by cancer biologists, investigations in other fields have uncovered exciting and often unpredicted roles for PI3K catalytic and regulatory subunits in normal cell function and in disease. Many of these functions impinge upon oncology by influencing the efficacy and toxicity of PI3K-targeted therapies. Here we provide a perspective on the roles of class I PI3Ks in the regulation of cellular metabolism and in immune system functions, two topics closely intertwined with cancer biology. We also discuss recent progress developing PI3K-targeted therapies for treatment of cancer and other diseases.

Sun J, Hopkins BD, Tsujikawa K, Perruzzi C, Adini I, Swerlick R, Bornstein P, Lawler J, Benjamin LE. Thrombospondin-1 modulates VEGF-A-mediated Akt signaling and capillary survival in the developing retina. Am J Physiol Heart Circ Physiol 296: H1344-H1351, 2009. First published March 20, 2009; doi: 10.1 152/ajpheart.01246.2008.-Microvascular development is often perceived to result from a balance of positive and negative factors that impact signaling for proliferation and survival. The survival signaling that results from hypoxia-induced VEGF-A has been well established, but the factors that antagonize this signaling have been poorly studied. As endogenous inhibitors of angiogenesis, thrombospondins (TSPs) are likely candidates to affekt survival signaling. Here we report that TSP! antagonized microvascular survival to retinal hyperoxia, and Akt signaling in both the retina and in cultured endothelial cells. TSP! expression is correlated with the association of the CD36 receptor with Src versus Fyn. In the presence of TSP1, CD36 is coprecipitated with Fyn as previously shown by others. However, in the absence of TSP1, there is a preferential association with Src. We now demonstrate that these Src family kinases play an important role in modulating microvascular survival in response to TSPI by crossing tsp1[sup-/-] mice to the src[sup-/-] and fyn[sup-/-] mice and testing the survival of retinal blood vessels in hyperoxia. We find that tsp1[sup-/-], fyn[sup-/-], and double-mutant tsp1[sup-/-]/fyn[sup-/-] mice have a similar enhancement of capillary survival in oxygen, whereas in a tsp[sup-/-] background the loss of only one allele of src restores the balance in survival and apoptosis to that of wild-type mice. Taken together, we hypothesize that TSP I antagonizes VEGF-driven Akt survival signaling in part through the recruitment of Fyn to membrane domains containing CD36, but when TSPI is absent, an opposing Src recruitment contributes to VEGF-driven Akt phosphorylation and capillary survival. [ABSTRACT FROM AUTHOR]

Background: The goal of precision oncology is to find effective therapeutics for every patient. Through the inclusion of emerging therapeutics in a high-throughput drug screening platform, our functional genomics pipeline inverts the common paradigm to identify patient populations that are likely to benefit from novel therapeutic strategies., Approach: Utilizing drug screening data across a panel of 46 cancer cell lines from 11 tumor lineages, we identified an ovarian cancer-specific sensitivity to the first-in-class CRL4 inhibitors KH-4-43 and 33-11. CRL4 (i.e., Cullin-4 RING E3 ubiquitin ligase) is known to be dysregulated in a variety of cancer contexts, making it an attractive therapeutic target. Unlike proteasome inhibitors that are associated with broad toxicity, CRL4 inhibition offers the potential for tumor-specific effects., Results: We observed that CRL4 inhibition negatively regulates core gene signatures that are upregulated in ovarian tumors and significantly slowed tumor growth as compared to the standard of care, cisplatin, in OVCAR8 xenografts. Building on this, we performed combination drug screening in conjunction with proteomic and transcriptomic profiling to identify ways to improve the antitumor effects of CRL4 inhibition in ovarian cancer models. CRL4 inhibition consistently resulted in activation of the mitogen-activated protein kinase (MAPK) signaling cascade at both the transcriptomic and protein levels, suggesting that survival signaling is induced in response to CRL4 inhibition. These observations were concordant with the results of the combination drug screens in seven ovarian cancer cell lines that showed CRL4 inhibition cooperates with MEK inhibition. Preclinical studies in OVCAR8 and A2780 xenografts confirmed the therapeutic potential of the combination of KH-4-43 and trametinib, which extended overall survival and slowed tumor progression relative to either single agent or the standard of care., Conclusions: Together, these data demonstrate the prospective utility of functional modeling pipelines for therapeutic development and underscore the clinical potential of CRL4 inhibition in the ovarian cancer context., Highlights: A precision medicine pipeline identifies ovarian cancer sensitivity to CRL4 inhibitors. CRL4 inhibition induces activation of MAPK signalling as identified by RNA sequencing, proteomics, and phosphoproteomics. Inhibitor combinations that target both CRL4 and this CRL4 inhibitor-induced survival signalling enhance ovarian cancer sensitivity to treatment., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

In this era of precision medicine, numerous workflows for the targeting of high-recurrent mutations in common tumor types have been developed, leaving patients with rare diseases with few options. Here, we implement a functional precision oncology approach utilizing comprehensive genomic profiling in combination with high-throughput drug screening, to identify tumor-specific drug sensitivities for patients with rare tumor types such as myxofibrosarcoma. From a patient with a high-grade myxofibrosarcoma, who was enrolled in the Englander Institute for Precision Medicine (EIPM) program, we established patient-derived 3D sarco - spheres and xenograft models for functional testing. In the absence of a large cohort of clinically similar cases, high-throughput drug screening was performed on the patient-derived cells, and compared with two other myxofibrosarcoma lines and a benign fibroblast line to functionally identify tumor-specific drug sensitivities. The addition of functional drug sensitivity testing to complement genomic profiling identified multiple therapeutic options that were further validated in patient derived xenograft models. Genomic analyses detected the frequently known codeletion of the tumor suppressors CDKN2A/B together with the methylthioadenosine phosphorylase ( MTAP ) and a TP53 E286fs*50 mutation. High-throughput drug screening demonstrated tumor-specific sensitivity to compounds targeting the cell cycle. Based on genomic analysis and high-throughput drug screening, we show that targeting the cell cycle in these tumors is a powerful approach. IMPLICATIONS: This study demonstrates the potential of functional testing to aid clinical decision making for patients with rare or molecularly complex malignancies when combined with comprehensive genomic profiling., (©2021 The Authors; Published by the American Association for Cancer Research.)