Notch signaling without the APH-2/Nicastrin subunit of Gamma Secretase in C. elegans germline stem cells.

The final step in Notch signaling activation is the transmembrane cleavage of Notch receptor by γ secretase. Thus far, genetic and biochemical evidence indicate that four subunits are essential for γ secretase activity in vivo: presenilin (the catalytic core), APH-1, PEN-2, and APH-2/Nicastrin. Although some γ secretase activity has been detected in APH-2/Nicastrin-deficient mammalian cell lines, the lack of biological relevance for this activity has left the quaternary γ secretase model unchallenged. Here we provide the first example of in vivo Notch signal transduction without APH-2/Nicastrin. The surprising dispensability of APH-2/Nicastrin is observed in C. elegans germline stem cells (GSCs), and contrasts with its essential role in previously described C. elegans Notch signaling events. Depletion of GLP-1/Notch, presenilin, APH-1, or PEN-2 causes a striking loss of GSCs. In contrast, aph-2/Nicastrin mutants maintain GSCs, and exhibit robust and localized expression of the downstream Notch target sygl-1. Interestingly, APH-2/Nicastrin is present in GSCs and becomes essential under conditions of compromised Notch function. Further insight is provided by reconstituting the C. elegans γ secretase complex in yeast, where we find that APH-2/Nicastrin increases, but is not essential for γ secretase activity. Together, our results are most consistent with a revised model of γ secretase in which the APH-2/Nicastrin subunit has a modulatory, rather than obligatory role. We propose that a trimeric presenilin-APH-1-PEN-2 γ secretase complex can provide a low level of γ secretase activity, and that cellular context determines whether or not APH-2/Nicastrin is essential for effective Notch signal transduction.

Re-education of myeloid immune cells to reduce regulatory T cell expansion and impede breast cancer progression.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. We demonstrate that NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the NLRP3 inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Loss of NR0B2 increased mammary tumor growth and metastasis. Small molecule agonists, including one developed here, reduced Treg expansion, reduced metastatic growth and improved the efficacy of ICB. This work identifies NR0B2 as a target to re-educate myeloid immune cells providing proof-of-principle that this cholesterol-homeostasis axis may have utility in enhancing ICB.

Degradation of GSPT1 causes TP53-independent cell death in leukemia while sparing normal hematopoietic stem cells.

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR/Cas9 screens implicated decreased translation initiation as protective following GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to the effects of GSPT1 degradation. We defined 2 Crbn amino acids that prevent Gspt1 degradation in mice, generated a knockin mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant acute myeloid leukemia.

The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K.

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.

Patterns of substrate affinity, competition, and degradation kinetics underlie biological activity of thalidomide analogs.

Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.

Crbn I391V is sufficient to confer in vivo sensitivity to thalidomide and its derivatives in mice.

Thalidomide and its derivatives, lenalidomide and pomalidomide, are clinically effective treatments for multiple myeloma and myelodysplastic syndrome with del(5q). These molecules lack activity in murine models, limiting investigation of their therapeutic activity or toxicity in vivo. Here, we report the development of a mouse model that is sensitive to thalidomide derivatives because of a single amino acid change in the direct target of thalidomide derivatives, cereblon (Crbn). In human cells, thalidomide and its analogs bind CRBN and recruit protein targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and subsequent degradation by the proteasome. We show that mice with a single I391V amino acid change in Crbn exhibit thalidomide-induced degradation of drug targets previously identified in human cells, including Ikaros (Ikzf1), Aiolos (Ikzf3), Zfp91, and casein kinase 1a1 (Ck1α), both in vitro and in vivo. We use the Crbn I391V model to demonstrate that the in vivo therapeutic activity of lenalidomide in del(5q) myelodysplastic syndrome can be explained by heterozygous expression of Ck1α in del(5q) cells. We found that lenalidomide acts on hematopoietic stem cells with heterozygous expression of Ck1α and inactivation of Trp53 causes lenalidomide resistance. We further demonstrate that Crbn I391V is sufficient to confer thalidomide-induced fetal loss in mice, capturing a major toxicity of this class of drugs. Further study of the Crbn I391V model will provide valuable insights into the in vivo efficacy and toxicity of this class of drugs.

Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome.

In historical attempts to treat morning sickness, use of the drug thalidomide led to the birth of thousands of children with severe birth defects. Despite their teratogenicity, thalidomide and related IMiD drugs are now a mainstay of cancer treatment; however, the molecular basis underlying the pleiotropic biology and characteristic birth defects remains unknown. Here we show that IMiDs disrupt a broad transcriptional network through induced degradation of several C2H2 zinc finger transcription factors, including SALL4, a member of the spalt-like family of developmental transcription factors. Strikingly, heterozygous loss of function mutations in SALL4 result in a human developmental condition that phenocopies thalidomide-induced birth defects such as absence of thumbs, phocomelia, defects in ear and eye development, and congenital heart disease. We find that thalidomide induces degradation of SALL4 exclusively in humans, primates, and rabbits, but not in rodents or fish, providing a mechanistic link for the species-specific pathogenesis of thalidomide syndrome.

The novel mechanism of lenalidomide activity.

Lenalidomide acts by a novel drug mechanism-modulation of the substrate specificity of the CRL4(CRBN) E3 ubiquitin ligase. In multiple myeloma, lenalidomide induces the ubiquitination of IKZF1 and IKZF3 by CRL4(CRBN). Subsequent proteasomal degradation of these transcription factors kills multiple myeloma cells. In del(5q) myelodysplastic syndrome, lenalidomide induces the degradation of CK1α, which preferentially affects del(5q) cells because they express this gene at haploinsufficient levels. In the future, modulation of ubiquitin ligase function may enable us to target previously "undruggable" proteins.

Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS.

Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)), resulting in CK1α degradation. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. We found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degradation of CK1α. We further demonstrate that minor side chain modifications in thalidomide and a novel analogue, CC-122, can modulate the spectrum of substrates targeted by CRL4(CRBN). These findings have implications for the clinical activity of lenalidomide and related compounds, and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.

Csnk1a1 inhibition has p53-dependent therapeutic efficacy in acute myeloid leukemia.

Despite extensive insights into the underlying genetics and biology of acute myeloid leukemia (AML), overall survival remains poor and new therapies are needed. We found that casein kinase 1 α (Csnk1a1), a serine-threonine kinase, is essential for AML cell survival in vivo. Normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected by shRNA-mediated knockdown of Csnk1a1. To identify downstream mediators of Csnk1a1 critical for leukemia cells, we performed an in vivo pooled shRNA screen and gene expression profiling. We found that Csnk1a1 knockdown results in decreased Rps6 phosphorylation, increased p53 activity, and myeloid differentiation. Consistent with these observations, p53-null leukemias were insensitive to Csnk1a1 knockdown. We further evaluated whether D4476, a casein kinase 1 inhibitor, would exhibit selective antileukemic effects. Treatment of leukemia stem cells (LSCs) with D4476 showed highly selective killing of LSCs over normal HSPCs. In summary, these findings demonstrate that Csnk1a1 inhibition causes reduced Rps6 phosphorylation and activation of p53, resulting in selective elimination of leukemia cells, revealing Csnk1a1 as a potential therapeutic target for the treatment of AML.

BRAF and MC1R in melanoma: different in head and neck tumors?

In this issue, Hacker et al. (2012) report the largest study to date on the association between MC1R variants and BRAF mutant melanoma. Although they did not observe a significant overall correlation, there was a significant negative association between BRAF and MC1R mutations for head/neck melanomas. This suggests a fundamental difference in pathogenesis between head/neck and truncal melanomas, which could contribute to their divergent prognoses.