Publications by authors named "Nathanael S Gray"

373 Publications

PRM-LIVE with Trapped Ion Mobility Spectrometry and Its Application in Selectivity Profiling of Kinase Inhibitors.

Anal Chem 2021 Oct 4;93(41):13791-13799. Epub 2021 Oct 4.

Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, United States.

Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.
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http://dx.doi.org/10.1021/acs.analchem.1c02349DOI Listing
October 2021

INK4 tumor suppressor proteins mediate resistance to CDK4/6 kinase inhibitors.

Cancer Discov 2021 Sep 20. Epub 2021 Sep 20.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center

Cyclin-dependent kinases 4 and 6 (CDK4/6), represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identify several genetic alterations (e.g. FAT1, PTEN or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g. p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C/D-cyclin/CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance.
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http://dx.doi.org/10.1158/2159-8290.CD-20-1726DOI Listing
September 2021

Correction: Fragment-based covalent ligand discovery.

RSC Chem Biol 2021 Apr 22;2(2):670-671. Epub 2021 Feb 22.

Department of Cancer Biology, Dana-Farber Cancer Institute Boston MA 02215 USA

[This corrects the article DOI: 10.1039/D0CB00222D.].
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http://dx.doi.org/10.1039/d1cb90008kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341680PMC
April 2021

Fragment-based covalent ligand discovery.

RSC Chem Biol 2021 Apr 9;2(2):354-367. Epub 2021 Feb 9.

Department of Cancer Biology, Dana-Farber Cancer Institute Boston MA 02215 USA

Targeted covalent inhibitors have regained widespread attention in drug discovery and have emerged as powerful tools for basic biomedical research. Fueled by considerable improvements in mass spectrometry sensitivity and sample processing, chemoproteomic strategies have revealed thousands of proteins that can be covalently modified by reactive small molecules. Fragment-based drug discovery, which has traditionally been used in a target-centric fashion, is now being deployed on a proteome-wide scale thereby expanding its utility to both the discovery of novel covalent ligands and their cognate protein targets. This powerful approach is allowing 'high-throughput' serendipitous discovery of cryptic pockets leading to the identification of pharmacological modulators of proteins previously viewed as "undruggable". The reactive fragment toolkit has been enabled by recent advances in the development of new chemistries that target residues other than cysteine including lysine and tyrosine. Here, we review the emerging area of covalent fragment-based ligand discovery, which integrates the benefits of covalent targeting and fragment-based medicinal chemistry. We discuss how the two strategies synergize to facilitate the efficient discovery of new pharmacological modulators of established and new therapeutic target proteins.
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http://dx.doi.org/10.1039/d0cb00222dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341086PMC
April 2021

Selective degradation-inducing probes for studying cereblon (CRBN) biology.

RSC Med Chem 2021 Aug 6;12(8):1381-1390. Epub 2021 Jul 6.

Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School Boston Massachusetts 02115 USA.

Targeted protein degradation represents a rapidly growing area in drug discovery and development. Moreover, small molecules that induce the targeted degradation of a given protein also represent an important addition to the chemical probes toolbox as these compounds can achieve selective protein knockdown, thus providing an approach that is orthogonal to genetic knockdowns. In order to develop degradation-inducing chemical probes for studying cereblon (CRBN) biology, we generated six CRBN-CRBN (homo-PROTAC) degraders and six CRBN-VHL (hetero-PROTAC) degraders. From these compounds we identified two potent and selective CRBN degraders ( and ), both of which are CRBN-VHL compounds. We characterized these lead degraders by quantitative proteomics in five cell lines (MM1.S, Kelly, SK-N-DZ, HEK293T, and MOLT-4) and observed high selectivity for CRBN in all cell lines. Furthermore, we directly compared our compounds to current lead CRBN degraders and demonstrated how these probes can be used as chemical knockdown reagents for studying CRBN-dependent processes. Overall, our work provides a roadmap for thorough degrader characterization by combination western and proteomic analysis, as illustrated by the identification of and as CRBN-knockdown tool compounds suitable for cell-based studies.
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http://dx.doi.org/10.1039/d0md00382dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8372211PMC
August 2021

Exploring Ligand-Directed -Acyl--alkylsulfonamide-Based Acylation Chemistry for Potential Targeted Degrader Development.

ACS Med Chem Lett 2021 Aug 21;12(8):1302-1307. Epub 2021 Jul 21.

Department of Chemical and Systems Biology, ChEM-H, Stanford Cancer Institute, School of Medicine, Stanford University, Stanford, California 94305, United States.

Ligand-directed bioconjugation strategies have been used for selective protein labeling in live cells or tissue samples in applications such as live-cell imaging. Here we hypothesized that a similar strategy could be used for targeted protein degradation. To test this possibility, we developed a series of CDK2-targeting -acyl--alkylsulfonamide (NASA)-containing acylation probes. The probes featured three components: a CDK2 homing ligand, a CRL4 E3 ligase recruiting ligand, and a NASA functionality. We determined that upon target binding, NASA-mediated reaction resulted in selective functionalization of Lys89 on purified or native CDK2. However, we were unable to observe CDK2 degradation, which is in contrast to the efficient degradation achieved by the use of a structurally similar reversible bivalent degrader. Our analysis suggests that the lack of degradation is due to the failure to form a productive CDK2:CRBN complex. Therefore, although this work demonstrates that NASA chemistry can be used for protein labeling, whether this strategy could enable efficient protein degradation remains an open question.
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http://dx.doi.org/10.1021/acsmedchemlett.1c00285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8366001PMC
August 2021

Targeting Pin1 renders pancreatic cancer eradicable by synergizing with immunochemotherapy.

Cell 2021 Sep 12;184(18):4753-4771.e27. Epub 2021 Aug 12.

Division of Translational Therapeutics, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA; Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.
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http://dx.doi.org/10.1016/j.cell.2021.07.020DOI Listing
September 2021

TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma.

Cancer Cell 2021 Sep 29;39(9):1262-1278.e7. Epub 2021 Jul 29.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; The Broad Institute of MIT and Harvard, Cambridge, MA, USA; Division of Pediatric Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA. Electronic address:

Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.
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http://dx.doi.org/10.1016/j.ccell.2021.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8443273PMC
September 2021

Dual targeting of salt inducible kinases and CSF1R uncouples bone formation and bone resorption.

Elife 2021 06 23;10. Epub 2021 Jun 23.

Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, United States.

Bone formation and resorption are typically coupled, such that the efficacy of anabolic osteoporosis treatments may be limited by bone destruction. The multi-kinase inhibitor YKL-05-099 potently inhibits salt inducible kinases (SIKs) and may represent a promising new class of bone anabolic agents. Here, we report that YKL-05-099 increases bone formation in hypogonadal female mice without increasing bone resorption. Postnatal mice with inducible, global deletion of SIK2 and SIK3 show increased bone mass, increased bone formation, and, distinct from the effects of YKL-05-099, increased bone resorption. No cell-intrinsic role of SIKs in osteoclasts was noted. In addition to blocking SIKs, YKL-05-099 also binds and inhibits CSF1R, the receptor for the osteoclastogenic cytokine M-CSF. Modeling reveals that YKL-05-099 binds to SIK2 and CSF1R in a similar manner. Dual targeting of SIK2/3 and CSF1R induces bone formation without concomitantly increasing bone resorption and thereby may overcome limitations of most current anabolic osteoporosis therapies.
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http://dx.doi.org/10.7554/eLife.67772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238509PMC
June 2021

The HCK/BTK inhibitor KIN-8194 is active in MYD88 driven lymphomas and overcomes mutated BTKCys481 ibrutinib resistance.

Blood 2021 06 16. Epub 2021 Jun 16.

Bing Center for Waldenstrom’s Macroglobulinemia, Dana-Farber Cancer Institute, Boston, MA.

Activating mutations in MYD88 promote malignant cell growth and survival through HCK mediated BTK activation. Ibrutinib binds to BTKCys481 and is active in B-cell malignancies driven by mutated MYD88. Mutations in BTKCys481 particularly BTKCys481Ser are common in patients with acquired ibrutinib resistance. We therefore performed an extensive medicinal chemistry campaign and identified KIN-8194 as a novel dual inhibitor of HCK and BTK. KIN-8194 showed potent and selective in vitro killing of MYD88 mutated lymphoma cells, including ibrutinib resistant BTKCys481Ser expressing cells. KIN-8194 demonstrated excellent bioavailability and pharmacokinetic parameters, with good tolerance in rodent models at pharmacologically achievable and active doses. Pharmacodynamic studies showed sustained HCK and BTK inhibition for 24 hours following single oral administration of KIN-8194 in MYD88 mutated TMD-8 ABC DLBCL xenografted mice with either wild-type BTK (BTKWT) or BTKCys481Ser expressing tumors. KIN-8194 showed superior survival benefit over ibrutinib in both BTKWT and BTKCys481Ser expressing TMD-8 DLBCL xenografted mice, including sustained complete responses >12 weeks off treatment in mice with BTKWT expressing TMD-8 tumors. The Bcl-2 inhibitor venetoclax enhanced the anti-tumor activity of KIN-8194 in BTKWT and BTKCys481Ser expressing MYD88 mutated lymphoma cells, and markedly reduced tumor growth and prolonged survival in mice with BTKCys481Ser expressing TMD-8 tumors treated with both drugs. The findings highlight the feasibility of targeting HCK, a key driver of mutated MYD88 pro-survival signaling, and provide a framework for the advancement of KIN-8194 for human studies in B-cell malignancies driven by HCK and BTK.
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http://dx.doi.org/10.1182/blood.2021011405DOI Listing
June 2021

The Cyclin-Dependent Kinase 8 (CDK8) Inhibitor DCA Promotes a Tolerogenic Chemical Immunophenotype in CD4 T Cells via a Novel CDK8-GATA3-FOXP3 Pathway.

Mol Cell Biol 2021 08 24;41(9):e0008521. Epub 2021 Aug 24.

Benaroya Research Institute, Seattle, Washington, USA.

Immune health requires innate and adaptive immune cells to engage precisely balanced pro- and anti-inflammatory forces. We employ the concept of chemical immunophenotypes to classify small molecules functionally or mechanistically according to their patterns of effects on primary innate and adaptive immune cells. The high-specificity, low-toxicity cyclin-dependent kinase 8 (CDK8) inhibitor 16-didehydro-cortistatin A (DCA) exerts a distinct tolerogenic profile in both innate and adaptive immune cells. DCA promotes regulatory T cells (T) and Th2 differentiation while inhibiting Th1 and Th17 differentiation in both murine and human cells. This unique chemical immunophenotype led to mechanistic studies showing that DCA promotes T differentiation in part by regulating a previously undescribed CDK8-GATA3-FOXP3 pathway that regulates early pathways of Foxp3 expression. These results highlight previously unappreciated links between T and Th2 differentiation and extend our understanding of the transcription factors that regulate T differentiation and their temporal sequencing. These findings have significant implications for future mechanistic and translational studies of CDK8 and CDK8 inhibitors.
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http://dx.doi.org/10.1128/MCB.00085-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384069PMC
August 2021

Acute pharmacological degradation of Helios destabilizes regulatory T cells.

Nat Chem Biol 2021 06 25;17(6):711-717. Epub 2021 May 25.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

The zinc-finger transcription factor Helios is critical for maintaining the identity, anergic phenotype and suppressive activity of regulatory T (T) cells. While it is an attractive target to enhance the efficacy of currently approved immunotherapies, no existing approaches can directly modulate Helios activity or abundance. Here, we report the structure-guided development of small molecules that recruit the E3 ubiquitin ligase substrate receptor cereblon to Helios, thereby promoting its degradation. Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of T cells, establishing a route towards Helios-targeting therapeutics. More generally, this study provides a framework for the development of small-molecule degraders for previously unligandable targets by reprogramming E3 ligase substrate specificity.
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http://dx.doi.org/10.1038/s41589-021-00802-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8162940PMC
June 2021

Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells.

Proteomics 2021 07 28;21(13-14):e2000098. Epub 2021 May 28.

Cancer Inflammation Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia.

Doublecortin-like kinase 1 (DCLK1) is a putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumors and a proposed driver gene for gastric cancer (GC). DCLK1 overexpression in a majority of solid cancers correlates with lymph node metastases, advanced disease and overall poor-prognosis. In cancer cells, DCLK1 expression has been shown to promote epithelial-to-mesenchymal transition (EMT), driving disruption of cell-cell adhesion, cell migration and invasion. Here, we report that DCLK1 influences small extracellular vesicle (sEV/exosome) biogenesis in a kinase-dependent manner. sEVs isolated from DCLK1 overexpressing human GC cell line MKN1 (MKN1 -sEVs), promote the migration of parental (non-transfected) MKN1 cells (MKN1 ). Quantitative proteome analysis of MKN1 -sEVs revealed enrichment in migratory and adhesion regulators (STRAP, CORO1B, BCAM, COL3A, CCN1) in comparison to MKN1 -sEVs. Moreover, using DCLK1-IN-1, a specific small molecule inhibitor of DCLK1, we reversed the increase in sEV size and concentration in contrast to other EV subtypes, as well as kinase-dependent cargo selection of proteins involved in EV biogenesis (KTN1, CHMP1A, MYO1G) and migration and adhesion processes (STRAP, CCN1). Our findings highlight a specific role of DCLK1-kinase dependent cargo selection for sEVs and shed new light on its role as a regulator of signaling in gastric tumorigenesis.
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http://dx.doi.org/10.1002/pmic.202000098DOI Listing
July 2021

Generation of a chemical genetic model for JAK3.

Sci Rep 2021 05 12;11(1):10093. Epub 2021 May 12.

Division of Cell Signalling and Immunology, School of Life Sciences, Wellcome Trust Building, University of Dundee, Dundee, DD1 5EH, UK.

Janus Kinases (JAKs) have emerged as an important drug target for the treatment of a number of immune disorders due to the central role that they play in cytokine signalling. 4 isoforms of JAKs exist in mammalian cells and the ideal isoform profile of a JAK inhibitor has been the subject of much debate. JAK3 has been proposed as an ideal target due to its expression being largely restricted to the immune system and its requirement for signalling by cytokine receptors using the common γ-chain. Unlike other JAKs, JAK3 possesses a cysteine in its ATP binding pocket and this has allowed the design of isoform selective covalent JAK3 inhibitors targeting this residue. We report here that mutating this cysteine to serine does not prevent JAK3 catalytic activity but does greatly increase the IC50 for covalent JAK3 inhibitors. Mice with a Cys905Ser knockin mutation in the endogenous JAK3 gene are viable and show no apparent welfare issues. Cells from these mice show normal STAT phosphorylation in response to JAK3 dependent cytokines but are resistant to the effects of covalent JAK3 inhibitors. These mice therefore provide a chemical-genetic model to study JAK3 function.
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http://dx.doi.org/10.1038/s41598-021-89356-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115619PMC
May 2021

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

Nat Chem Biol 2021 09 10;17(9):954-963. Epub 2021 May 10.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.
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http://dx.doi.org/10.1038/s41589-021-00786-7DOI Listing
September 2021

Structure-activity relationship study of THZ531 derivatives enables the discovery of BSJ-01-175 as a dual CDK12/13 covalent inhibitor with efficacy in Ewing sarcoma.

Eur J Med Chem 2021 Oct 20;221:113481. Epub 2021 Apr 20.

Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA. Electronic address:

Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.
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http://dx.doi.org/10.1016/j.ejmech.2021.113481DOI Listing
October 2021

Inhibition of CDK4/6 Promotes CD8 T-cell Memory Formation.

Cancer Discov 2021 Oct 3;11(10):2564-2581. Epub 2021 May 3.

Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts.

CDK4/6 inhibitors are approved to treat breast cancer and are in trials for other malignancies. We examined CDK4/6 inhibition in mouse and human CD8 T cells during early stages of activation. Mice receiving tumor-specific CD8 T cells treated with CDK4/6 inhibitors displayed increased T-cell persistence and immunologic memory. CDK4/6 inhibition upregulated MXD4, a negative regulator of MYC, in both mouse and human CD8 T cells. Silencing of in mouse CD8 T cells demonstrated the importance of this axis for memory formation. We used single-cell transcriptional profiling and T-cell receptor clonotype tracking to evaluate recently activated human CD8 T cells in patients with breast cancer before and during treatment with either palbociclib or abemaciclib. CDK4/6 inhibitor therapy in humans increases the frequency of CD8 memory precursors and downregulates their expression of MYC target genes, suggesting that CDK4/6 inhibitors in patients with cancer may augment long-term protective immunity. SIGNIFICANCE: CDK4/6 inhibition skews newly activated CD8 T cells toward a memory phenotype in mice and humans with breast cancer. CDK4/6 inhibitors may have broad utility outside breast cancer, particularly in the neoadjuvant setting to augment CD8 T-cell priming to tumor antigens prior to dosing with checkpoint blockade..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8487897PMC
October 2021

Discovery of a Potent Degrader for Fibroblast Growth Factor Receptor 1/2.

Angew Chem Int Ed Engl 2021 07 14;60(29):15905-15911. Epub 2021 Jun 14.

Department of Chemical and Systems Biology, Chem-H and Stanford Cancer Institute, Stanford School of Medicine, Stanford University, Stanford, CA, USA.

Aberrant activation of FGFR signaling occurs in many cancers, and ATP-competitive FGFR inhibitors have received regulatory approval. Despite demonstrating clinical efficacy, these inhibitors exhibit dose-limiting toxicity, potentially due to a lack of selectivity amongst the FGFR family and are poorly tolerated. Here, we report the discovery and characterization of DGY-09-192, a bivalent degrader that couples the pan-FGFR inhibitor BGJ398 to a CRL2 E3 ligase recruiting ligand, which preferentially induces FGFR1&2 degradation while largely sparing FGFR3&4. DGY-09-192 exhibited two-digit nanomolar DC s for both wildtype FGFR2 and several FGFR2-fusions, resulting in degradation-dependent antiproliferative activity in representative gastric cancer and cholangiocarcinoma cells. Importantly, DGY-09-192 induced degradation of a clinically relevant FGFR2 fusion protein in a xenograft model. Taken together, we demonstrate that DGY-09-192 has potential as a prototype FGFR degrader.
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http://dx.doi.org/10.1002/anie.202101328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324087PMC
July 2021

Discovery and resistance mechanism of a selective CDK12 degrader.

Nat Chem Biol 2021 06 22;17(6):675-683. Epub 2021 Mar 22.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. In the present study, we report the rational design and characterization of a CDK12-specific degrader, BSJ-4-116. BSJ-4-116 selectively degraded CDK12 as assessed through quantitative proteomics. Selective degradation of CDK12 resulted in premature cleavage and poly(adenylation) of DDR genes. Moreover, BSJ-4-116 exhibited potent antiproliferative effects, alone and in combination with the poly(ADP-ribose) polymerase inhibitor olaparib, as well as when used as a single agent against cell lines resistant to covalent CDK12 inhibitors. Two point mutations in CDK12 were identified that confer resistance to BSJ-4-116, demonstrating a potential mechanism that tumor cells can use to evade bivalent degrader molecules.
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http://dx.doi.org/10.1038/s41589-021-00765-yDOI Listing
June 2021

Targeting oncoproteins with a positive selection assay for protein degraders.

Sci Adv 2021 Feb 5;7(6). Epub 2021 Feb 5.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal ("down") assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal ("up") assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9-based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.
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http://dx.doi.org/10.1126/sciadv.abd6263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864573PMC
February 2021

Targeted brachyury degradation disrupts a highly specific autoregulatory program controlling chordoma cell identity.

Cell Rep Med 2021 Jan 19;2(1):100188. Epub 2021 Jan 19.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop.
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http://dx.doi.org/10.1016/j.xcrm.2020.100188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817874PMC
January 2021

Discovery of a Pyrimidothiazolodiazepinone as a Potent and Selective Focal Adhesion Kinase (FAK) Inhibitor.

ACS Med Chem Lett 2021 Jan 14;12(1):30-38. Epub 2020 Dec 14.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, United States.

Focal adhesion kinase (FAK) is a tyrosine kinase with prominent roles in protein scaffolding, migration, angiogenesis, and anchorage-independent cell survival and is an attractive target for the development of cancer therapeutics. However, current FAK inhibitors display dual kinase inhibition and/or significant activity on several kinases. Although multitargeted activity is at times therapeutically advantageous, such behavior can also lead to toxicity and confound chemical-biology studies. We report a novel series of small molecules based on a tricyclic pyrimidothiazolodiazepinone core that displays both high potency and selectivity for FAK. Structure-activity relationship (SAR) studies explored modifications to the thiazole, diazepinone, and aniline "tail," which identified lead compound BJG-03-025. BJG-03-025 displays potent biochemical FAK inhibition (IC = 20 nM), excellent kinome selectivity, activity in 3D-culture breast and gastric cancer models, and favorable pharmacokinetic properties in mice. BJG-03-025 is a valuable chemical probe for evaluation of FAK-dependent biology.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7812608PMC
January 2021

The Kinase Chemogenomic Set (KCGS): An Open Science Resource for Kinase Vulnerability Identification.

Int J Mol Sci 2021 Jan 8;22(2). Epub 2021 Jan 8.

Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.
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http://dx.doi.org/10.3390/ijms22020566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826789PMC
January 2021

Mammalian cell proliferation requires noncatalytic functions of O-GlcNAc transferase.

Proc Natl Acad Sci U S A 2021 01;118(4)

Department of Microbiology, Blavatnik Institute of Harvard Medical School, Boston, MA 02115;

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT's myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT's enzymatic activities for cell viability. Using genetic complementation, we found that OGT's glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT's catalytic and noncatalytic activities affect protein abundance, we found that OGT's noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.
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http://dx.doi.org/10.1073/pnas.2016778118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848692PMC
January 2021

An Embryonic Diapause-like Adaptation with Suppressed Myc Activity Enables Tumor Treatment Persistence.

Cancer Cell 2021 02 7;39(2):240-256.e11. Epub 2021 Jan 7.

Department of Data Sciences, Dana-Farber Cancer Institute & Harvard T.H. Chan School of Public Health, Boston, MA, USA.

Treatment-persistent residual tumors impede curative cancer therapy. To understand this cancer cell state we generated models of treatment persistence that simulate the residual tumors. We observe that treatment-persistent tumor cells in organoids, xenografts, and cancer patients adopt a distinct and reversible transcriptional program resembling that of embryonic diapause, a dormant stage of suspended development triggered by stress and associated with suppressed Myc activity and overall biosynthesis. In cancer cells, depleting Myc or inhibiting Brd4, a Myc transcriptional co-activator, attenuates drug cytotoxicity through a dormant diapause-like adaptation with reduced apoptotic priming. Conversely, inducible Myc upregulation enhances acute chemotherapeutic activity. Maintaining residual cells in dormancy after chemotherapy by inhibiting Myc activity or interfering with the diapause-like adaptation by inhibiting cyclin-dependent kinase 9 represent potential therapeutic strategies against chemotherapy-persistent tumor cells. Our study demonstrates that cancer co-opts a mechanism similar to diapause with adaptive inactivation of Myc to persist during treatment.
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http://dx.doi.org/10.1016/j.ccell.2020.12.002DOI Listing
February 2021

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Cell Rep 2021 01;34(1):108532

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.
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http://dx.doi.org/10.1016/j.celrep.2020.108532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8485877PMC
January 2021

Mapping the Degradable Kinome Provides a Resource for Expedited Degrader Development.

Cell 2020 12 3;183(6):1714-1731.e10. Epub 2020 Dec 3.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.
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http://dx.doi.org/10.1016/j.cell.2020.10.038DOI Listing
December 2020

Discovery of a Selective, Covalent IRAK1 Inhibitor with Antiproliferative Activity in MYD88 Mutated B-Cell Lymphoma.

ACS Med Chem Lett 2020 Nov 9;11(11):2238-2243. Epub 2020 Oct 9.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, United States.

Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating the innate immune response against foreign pathogens. Additionally, dysregulation of IRAK1 signaling plays a role in neoplastic disorders. For example, IRAK1 was shown to be important for survival and proliferation in many B-cell lymphomas, including Waldenström's macroglobulinemia (WM) and ABC subtype Diffused Large B-cell Lymphoma (DLBCL) cells. Here, we report the discovery of a highly potent and selective covalent inhibitor of IRAK1, JH-X-119-01. Intact protein MS labeling studies confirmed that JH-X-119-01 irreversibly labels IRAK1 at C302. This compound exhibited cytotoxic activity at single digit micromolar concentrations in a panel of WM, DLBCL, and lymphoma cell lines expressing MYD88. Cotreatment of JH-X-119-01 with the BTK inhibitor ibrutinib resulted in synergistic killing effects in these systems. Taken together, JH-X-119-01 represents a highly selective probe of IRAK1 for further development.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667833PMC
November 2020

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

J Clin Invest 2020 11;130(11):5875-5892

Division of Cancer Therapeutics, Institute of Cancer Research (ICR), London and Royal Marsden NHS Trust, Sutton, United Kingdom.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.
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http://dx.doi.org/10.1172/JCI134132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598076PMC
November 2020
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