Search

Supplementary Table 2

Supplementary Figures S1-S11

Colorectal cancer is driven by mutations that activate canonical WNT/β-catenin signaling, but inhibiting WNT has significant on-target toxicity, and there are no approved therapies targeting dominant oncogenic drivers. We recently found that activating a β-catenin–independent branch of WNT signaling that inhibits GSK3-dependent protein degradation induces asparaginase sensitivity in drug-resistant leukemias. To test predictions from our model, we turned to colorectal cancer because these cancers can have WNT-activating mutations that function either upstream (i.e., R-spondin fusions) or downstream (APC or β-catenin mutations) of GSK3, thus allowing WNT/β-catenin and WNT-induced asparaginase sensitivity to be unlinked genetically. We found that asparaginase had little efficacy in APC or β-catenin–mutant colorectal cancer, but was profoundly toxic in the setting of R-spondin fusions. Pharmacologic GSK3α inhibition was sufficient for asparaginase sensitization in APC or β-catenin–mutant colorectal cancer, but not in normal intestinal progenitors. Our findings demonstrate that WNT-induced therapeutic vulnerabilities can be exploited for colorectal cancer therapy.Significance:Solid tumors are thought to be asparaginase-resistant via de novo asparagine synthesis. In leukemia, GSK3α-dependent protein degradation, a catabolic amino acid source, mediates asparaginase resistance. We found that asparaginase is profoundly toxic to colorectal cancers with WNT-activating mutations that inhibit GSK3. Aberrant WNT activation can provide a therapeutic vulnerability in colorectal cancer.See related commentary by Davidsen and Sullivan, p. 1632.This article is highlighted in the In This Issue feature, p. 1611

CSNK1A1 is a serine/threonine kinase involved in multiple cellular processes, including cell division, beta catenin signaling, and TP53 activation. Inhibition of CSNK1A1 has previously been validated as a therapeutic strategy in hematologic malignancy, and degradation of CSNK1A1 protein is the downstream mechanism of action for lenalidomide in 5q- myelodysplasia (Krönke, et al. Nature. 2015.). However, lenalidomide is inactive in most solid tumor models, thus limiting the study of CSNK1A1 inhibition in other contexts. Analysis of genetic loss-of-function data from the Cancer Dependency Map reveals multiple sensitive models, including lineage-specific enrichment in colorectal and gastric cancer. In an academic-industry collaboration, we a) developed first-in-class potent and selective ATP-competitive CSNK1A1 small molecule inhibitors with preclinical anti-cancer efficacy in vivo, and b) identified FAM83 expression as a key determinant of inhibitor sensitivity. We identified a tetrahydro-pyrrolopyridinone scaffold that was subsequently optimized to yield BAY-888 (CSNK1A1 IC50 4 nM @ 10 μM ATP; 63 nM @ 1 mM ATP) and BAY-204 (CSNK1A1 IC50 2 nM @ 10 μM ATP; 12 nM @ 1 mM ATP). The crystal structure of CSNK1A1 in complex with BAY-888 confirmed compound binding in the ATP binding pocket. Across the PRISM barcoded cell line panel of more than 500 solid tumor cell lines, inhibitors phenocopy the CSNK1A1 shRNA knockdown profile. To determine downstream mediators of CSNK1A1 inhibitor sensitivity, we performed co-IP mass spectrometry following CSNK1A1 pulldown and global phosphoproteomic assays following inhibitor treatment. We identified multiple interacting proteins that are also phosphorylation targets, including FAM83 family members. FAM83 was recently reported to mediate the subcellular localization of CSNK1A1 (Fulcher, et al. Sci Signal. 2018.). Excitingly, the baseline expression of FAM83B and FAM83H correlates with inhibitor and shRNA cell line sensitivity. Modulation of FAM83H expression altered CSNK1A1 localization and sensitivity to CSNK1A1 inhibition. BAY-888 and BAY-204 are orally bioavailable and were evaluated in multiple murine cell line xenograft models. We observed promising efficacy in DLBCL (TMD8) in vivo as well as in multiple FAM83-high solid tumor models, including colorectal (HCT116 and HT29), gastric (IM95), and urothelial cancer (KU19-19). We identified RPS6 phosphorylation as one of the PD biomarkers correlating with efficacy in vivo. In summary, CSNK1A1 is a promising target with anti-tumor efficacy and achievable therapeutic index in preclinical models of FAM83-high solid tumors. Citation Format: Steven M. Corsello, Huajia Zhang, Rajesha Rupaimoole, Volker K. Schulze, Clara Lemos, Kasia B. Handing, Douglas L. Orsi, Mrinal Shekhar, Ulrike Sack, Sven Christian, Wilhelm Bone, Ranad Humeidi, William Colgan, Stephanie Hoyt, Andrew Cherniack, Jens Schroder, Stefan Kaulfuss, Krzysztof Brzezinka, Oliver von Ahsen, Anne Mengel, Roman C. Hillig, Detlev Suelzle, Jeremie Mortier, Caitlin Harrington, Rohith Nagari, Justyna Wierzbinska, Derek Chiang, Georg Beckmann, Meagan Olive, Namrata Udeshi, Annie Apffel, Steven Carr, Philip Lienau, Christian Lechner, Ulf Boemer, Alisha Caliman, David McKinney, Florence Wagner, Dominik Mumberg, Marcus Bauser, Andrea Haegebarth, Knut Eis, Ashley Eheim, Todd R. Golub. Discovery of potent and selective CSNK1A1 inhibitors for solid tumor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3588.

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk−, with the complexes failing to associate with chromosomes.

The Escherichia coli SMC complex, MukBEF, acts in chromosome segregation. MukBEF shares the distinctive architecture of other SMC complexes, with one prominent difference; unlike other kleisins, MukF forms dimers through its N-terminal domain. We show that a 4-helix bundle adjacent to the MukF dimerisation domain interacts functionally with the MukB coiled-coiled ‘neck’ adjacent to the ATPase head. We propose that this interaction leads to an asymmetric tripartite complex, as in other SMC complexes. Since MukF dimerisation is preserved during this interaction, MukF directs the formation of dimer of dimer MukBEF complexes, observed previously in vivo. The MukF N- and C-terminal domains stimulate MukB ATPase independently and additively. We demonstrate that impairment of the MukF interaction with MukB in vivo leads to ATP hydrolysis-dependent release of MukBEF complexes from chromosomes., eLife digest Most DNA in a cell is arranged in structures called chromosomes. From bacteria to humans, chromosomes have to be compacted and highly organized to allow the cells to maintain and use their genetic information. In all organisms, large ring-shaped protein complexes play a crucial role in managing chromosomes. They transport and organize DNA thanks to reactions whose precise mechanism remains unknown. In bacteria, MukB and a type of kleisin called MukF are two examples of molecules involved in chromosome management. Two MukBs join at one end to form a hinge; at the other end, each MukB protein has a neck and a head. The two heads are linked by the kleisin to form a large protein ring, which can open to capture DNA. The MukB heads can trigger a biochemical reaction that creates the energy essential to trap and release DNA during DNA transport. Here, Zawadzka et al. study how the different components of the MukB-kleisin complex interact with each other to undergo the biochemical reactions that lead to DNA transport. The experiments show that the kleisin joins two MukB heads by attaching the base of one to the neck of the other, asymmetrically closing the ring. The separate interactions of different regions of the kleisin to the head and neck of MukB independently activate the two MukB heads, thereby controlling essential steps in the reactions with DNA. Two MukB-kleisin ring complexes are joined to each other because of a tight interaction between the two kleisin molecules. This leads Zawadzka et al. to suggest that DNA is sequentially grabbed and released from these two rings during DNA transport, similar to how a climbing rope is attached and released through carabiners. Cells cannot survive or be healthy without their chromosomes being accurately managed. It is still unclear how molecules such as MukBs and kleinsins drive this process. A better picture of their structure and interactions is an essential first step to understand these mechanisms.

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk−, with the complexes failing to associate with chromosomes.